Objective To explore the protective effect and possible mechanism of modified Shenqi Dihuang Decoction on podocytes in db/db mice based on ROS/NLRP3/GSDMD signaling pathway.Methods Fifty 8-week-old male db/db mice(SPF grade)were randomly divided into the model group,losartan group and TCM low-,medium-and high-dosage groups,with 10 mice in each group.Ten heterozygous db/m mice served as the blank group.Interventions were administered respectively for 12 weeks.The body mass,random blood glucose,serum creatinine,blood urea nitrogen and 24 h urinary protein content were detected,HE,PAS,PASM,Masson and Sirius red staining was used to observe the morphology of renal tissue,transmission electron microscopy was used to observe the ultrastructure of renal tissue,fluorescent probes were used to observe the release of ROS in renal tissue,immunofluorescence staining was used to detect the expression of Nephrin,NLRP3,Cleaved Caspase-1 and GSDMD-N in renal tissue,and Western blot was used to detect the expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18,Nephrin,Podocin,PODXL,WT-1 and Desmin proteins.Results Compared with the blank group,the body mass and random blood glucose of the model group mice significantly increased(P<0.05),and the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein were significantly increased(P<0.05);glomerular hypertrophy,dilation of renal glomeruli and tubules,thickening of basement membrane,matrix proliferation in mesangial area,abnormal deposition of collagen fibers in renal interstitium,accompanied by damage to renal tubular epithelial structure and focal glomerulosclerosis,significant increase in type Ⅰ collagen deposition,extensive fusion of podocyte processes,and scattered electron dense material in the basement membrane or subepithelial layer;the ROS content in renal tissue significantly increased(P<0.05),and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin significantly increased(P<0.05),the protein expression of Nephrin,Podocin,PODXL and WT1 significantly decreased(P<0.05).Compared with the model group,the body mass and random blood glucose of mice in each dosage of TCM group were relatively stable,the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein decreased;the pathological damage to renal tissue was reduced,the ultrastructure of podocytes was improved,and the density of podocytes increased;the ROS content decreased,and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin decreased,while the protein expression of Nephrin,Podocin,WT1 and PODXL increased.With the dosage of modified Shenqi Dihuang Decoction increased,the improvement effect gradually strengthened,and the differences in TCM high-dose group was statistically significant(P<0.05).Conclusion Modified Shenqi Dihuang Decoction can protects podocytes in db/db mice,potentially by modulating the ROS/NLRP3/GSDMD signaling pathway.

BACKGROUND:Spinal osteosarcoma is a rare and highly aggressive malignant tumor.Most existing studies are based on small sample sizes and have inconsistent results,making it difficult to provide reliable clinical guidance.Especially in China,due to the low incidence of spinal osteosarcoma and limited related research,clinicians lack effective prognostic tools during treatment.OBJECTIVE:To construct and validate a nomogram model for predicting the survival of spinal osteosarcoma patients based on the Surveillance,Epidemiology,and End Results(SEER)database,providing scientific evidence for clinical decision-making,particularly for optimizing treatment plans for Chinese patients.METHODS:This study conducted a retrospective analysis of patient data diagnosed with spinal osteosarcoma from the SEER database between 2000 and 2021.First,independent prognostic factors associated with specific mortality from spinal osteosarcoma were identified through univariate and multivariate Cox proportional hazards models.Subsequently,these independent prognostic factors were used to construct a nomogram model for predicting survival rates of spinal osteosarcoma patients using the"rms"package in RStudio.The model's discrimination was assessed using the C-index.Predictive ability was validated through receiver operating characteristic curves and area under the curve values.Calibration was evaluated by calibration plots,and clinical value was measured using decision curve analysis.Additionally,Kaplan-Meier survival analysis was performed to assess the rationality of the nomogram groupings.RESULTS AND CONCLUSION:(1)The final model included six variables:chemotherapy,tumor size,histological type,grade,race,and surgical intervention.(2)The C-indices of the model in the training and validation sets were 0.685 and 0.673,respectively,indicating good discrimination.(3)Calibration curves showed high consistency between predicted survival probabilities and actual survival probabilities.(4)Decision curve analysis indicated that the model provided significant net benefits across a wide range of mortality risks.(5)Kaplan-Meier survival analysis revealed significant differences in prognosis between high-risk and low-risk groups.(6)The constructed nomogram model accurately predicts the 1-year,2-year,and 3-year survival rates of spinal osteosarcoma patients,demonstrating high clinical applicability.This model not only provides an effective survival prediction tool for American patients but also offers important insights for optimizing treatment plans for spinal osteosarcoma patients in China.Future research should further validate the model's applicability in different populations and explore the impact of novel treatment methods on the prognosis of spinal osteosarcoma,aiming to improve the survival rates and quality of life of patients in China.

Objective To construct a clinical-radiological nomo-gram model for severe mycoplasma pneumoniae pneumonia coinfec-tion with other pathogens(Co-SMPP)in children.Methods The clinical and radiological data of children with severe mycoplasma pneumoniae pneumonia(SMPP)who underwent nucleic acid testing or bronchoalveolar lavage(BAL)were analyzed retrospectively.The data analysis were performed by using SPSS 27.0 software.The group comparison between simple SMPP and Co-SMPP children was conducted by using t-tests,Mann-Whitney U tests,or chi-square tests.Nomogram analysis was performed by using R software and rms packages.The predictive performance of the model was evaluated by using the receiver operating characteristic(ROC)curve.Results A total of 194 SMPP children were included in the study,including 136 cases(70.1％)with simple SMPP,58 cases(29.9％)with Co-SMPP.The fibrinogen and albumin levels were lower in Co-SMPP children[(3.53±0.85)g/L,41.00(39.03,43.68)g/L]than in simple SMPP children[(3.79±0.80)g/L,42.80(41.00,44.40)g/L],with P values of 0.047 and 0.036,respec-tively.The probability of bronchial stenosis and grid shadow were higher in Co-SMPP children than in simple SMPP children,and there were significant differences between the two groups(P＜0.001,P=0.010).The odds ratio of bronchial stenosis in predicting Co-SMPP children was 14.085.The clinical-radiological nomogram model had an area under the curve(AUC)of 0.840,with sensi-tivity and specificity of 0.756 and 0.848,respectively.Conclusion The nomogram model based on clinical-radiological features can effectively predict Co-SMPP.

BACKGROUND:Spinal osteosarcoma is a rare and highly aggressive malignant tumor.Most existing studies are based on small sample sizes and have inconsistent results,making it difficult to provide reliable clinical guidance.Especially in China,due to the low incidence of spinal osteosarcoma and limited related research,clinicians lack effective prognostic tools during treatment.OBJECTIVE:To construct and validate a nomogram model for predicting the survival of spinal osteosarcoma patients based on the Surveillance,Epidemiology,and End Results(SEER)database,providing scientific evidence for clinical decision-making,particularly for optimizing treatment plans for Chinese patients.METHODS:This study conducted a retrospective analysis of patient data diagnosed with spinal osteosarcoma from the SEER database between 2000 and 2021.First,independent prognostic factors associated with specific mortality from spinal osteosarcoma were identified through univariate and multivariate Cox proportional hazards models.Subsequently,these independent prognostic factors were used to construct a nomogram model for predicting survival rates of spinal osteosarcoma patients using the"rms"package in RStudio.The model's discrimination was assessed using the C-index.Predictive ability was validated through receiver operating characteristic curves and area under the curve values.Calibration was evaluated by calibration plots,and clinical value was measured using decision curve analysis.Additionally,Kaplan-Meier survival analysis was performed to assess the rationality of the nomogram groupings.RESULTS AND CONCLUSION:(1)The final model included six variables:chemotherapy,tumor size,histological type,grade,race,and surgical intervention.(2)The C-indices of the model in the training and validation sets were 0.685 and 0.673,respectively,indicating good discrimination.(3)Calibration curves showed high consistency between predicted survival probabilities and actual survival probabilities.(4)Decision curve analysis indicated that the model provided significant net benefits across a wide range of mortality risks.(5)Kaplan-Meier survival analysis revealed significant differences in prognosis between high-risk and low-risk groups.(6)The constructed nomogram model accurately predicts the 1-year,2-year,and 3-year survival rates of spinal osteosarcoma patients,demonstrating high clinical applicability.This model not only provides an effective survival prediction tool for American patients but also offers important insights for optimizing treatment plans for spinal osteosarcoma patients in China.Future research should further validate the model's applicability in different populations and explore the impact of novel treatment methods on the prognosis of spinal osteosarcoma,aiming to improve the survival rates and quality of life of patients in China.

Objective To construct a clinical-radiological nomo-gram model for severe mycoplasma pneumoniae pneumonia coinfec-tion with other pathogens(Co-SMPP)in children.Methods The clinical and radiological data of children with severe mycoplasma pneumoniae pneumonia(SMPP)who underwent nucleic acid testing or bronchoalveolar lavage(BAL)were analyzed retrospectively.The data analysis were performed by using SPSS 27.0 software.The group comparison between simple SMPP and Co-SMPP children was conducted by using t-tests,Mann-Whitney U tests,or chi-square tests.Nomogram analysis was performed by using R software and rms packages.The predictive performance of the model was evaluated by using the receiver operating characteristic(ROC)curve.Results A total of 194 SMPP children were included in the study,including 136 cases(70.1％)with simple SMPP,58 cases(29.9％)with Co-SMPP.The fibrinogen and albumin levels were lower in Co-SMPP children[(3.53±0.85)g/L,41.00(39.03,43.68)g/L]than in simple SMPP children[(3.79±0.80)g/L,42.80(41.00,44.40)g/L],with P values of 0.047 and 0.036,respec-tively.The probability of bronchial stenosis and grid shadow were higher in Co-SMPP children than in simple SMPP children,and there were significant differences between the two groups(P＜0.001,P=0.010).The odds ratio of bronchial stenosis in predicting Co-SMPP children was 14.085.The clinical-radiological nomogram model had an area under the curve(AUC)of 0.840,with sensi-tivity and specificity of 0.756 and 0.848,respectively.Conclusion The nomogram model based on clinical-radiological features can effectively predict Co-SMPP.

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.

Radiomics represents a non-invasive, quantitative image analysis method, aimed at correlating high-throughput quantitative radiomics features with clinical and biological endpoints. This method enables comprehensive, multiple, and non-invasive tumor assessment based on different types of images in various stages of radiotherapy. Advancements have been made in the applications of radiomics combined with machine learning-based statistical method in multiple fields including individualized target delineation, the prediction of local recurrence and radiation-induced injuries, the assessment of distant metastasis risks, the prediction of tumor motion, adaptive radiotherapy (ART), and multi-omics analysis. These advancements provide more possibilities for personalized radiotherapy. This study presents a brief introduction to the applications of radiomics in personalized radiotherapy.

[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

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.