The Role And Mechanisms Of M1 Macrophages Polarization In Promoting The Destruction Of Vertebral Body And Intervertebral Disc Cells In Spinal Tuberculosis

Objective To investigate and analyze the role and mechanisms of M1 macrophages polarization in promoting the destruction of vertebral body and intervertebral disc cells in spinal tuberculosis.Method We found macrophage polarization changes through bioinformatics analysis of GEO dataset and verified through flow cytometry.Then we found the differentially expressed genes after macrophage polarization and verified with q RT-PCR and Western blot.its role in diagnostic ability,therapeutic effect evaluation,drug resistance and bone destruction were analyzed with multiple data.Then,the process of osteoclast differentiation was intervened by si RNA to observe its effect on osteoclast differentiation.Result Bioinformatics analysis showed that the monocytes to lymphoids ratio(MLR)in the tuberculosis group was significantly higher.And 326 patients we collected verified the above results.The results of bioinformatics analysis also showed that macrophages in TB group were significantly polarized to M1 type.Moreover,the expression of ferroptosis related SOCS1 increased significantly after macrophages were significantly polarized to M1 type.SOCS1 has certain diagnostic ability for tuberculosis,and the expression decreased significantly after patients received standard anti-TB treatment.Bioinformatics analysis also found that SOCS1 was positive correlation with key molecule in osteoclast differentiation NF-κB1,NF-κB2 and NFATC1.Subsequent intervention experiments confirmed that while the expression of SOCS1 gene was inhibited,the key molecule NF-κB1,NF-κB2 and NFATC1 also decreased significantly,and osteoclast differentiation was significantly inhibited.In addition,the results of q RT-PCR and immunohistochemistry showed that there was a negative correlation between the expression of ferroptosis related gene SOCS1 and ferroptosis key molecule GPX4 in intervertebral disc tissue.Conclusion The macrophages were M1 type polarization and the expression of SOCS1 is up-regulated after M1 macrophage polarization in spinal TB patients.SOCS1 promotes osteoclast differentiation by activating NF-κB1/NFATC1 leading to the destruction of bone tissue.In the intervertebral disc tissue of spinal tuberculosis patients,SOCS1 may induce the occurrence of ferroptosis in intervertebral disc cells of spinal tuberculosis patients by inhibiting the expression of GPX4,resulting in the destruction of intervertebral disc.Part 1 Clinical hematologic changes in patients with spinal tuberculosisObjective This study is aimed to develop nomogram the diagnosis of osteoarticular tuberculosis(TB)by clinical hematological indicators.Method We downloaded the expression profile of GSE83456 from GEO and proceed x Cell score estimation to obtained the immune cell type abundance scores.The routine blood examinations of 326 patients were collected for further validation.We analyzed univariate and multivariate logistic regression to identified independent predicted factor for developing the nomogram.The performance of the nomogram was assessed using the receiver operating characteristic(ROC)curves.The correlation of erythrocyte sedimentation rate(ESR)with lymphocytes,monocytes,and monocytes to lymphoids ratio(MLR)was performed and visualized in osteoarticular TB patients.Result Compared with the healthy control group in the dataset GSE83456,the x Cell score of basophils,monocytes,neutrophils,and platelets was higher,while lymphoid was lower in the extrapulmonary tuberculosis(EPTB)group.The clinical data showed that the cell count of monocytes were much higher,while the cell counts of lymphocytes were lower in the spinal TB group.The area under the curve(AUC)for osteoarticular TB diagnosis of the MLR in dataset GSE83456 was 0.798,and 0.737 for the clinical data.We identified the MLR,Body Mass Index(BMI),and ESR as the independent predictive factors for spinal TB diagnosis and constructed a nomogram for the clinical diagnosis of spinal TB.AUCs of this nomogram was 0.843.Conclusion We demonstrated a significant upregulated of MLR in the EPTB compared with the non-TB patients.Moreover,we constructed a nomogram for the clinical diagnosis of the spinal TB diagnosis,which works satisfactorily.Part 2 Detection of macrophage polarization and the expression of SOCS1 in patients with spinal TBObjective To investigate and analyze the relationship between macrophage polarization and the expression of suppressor of cytokine signaling 1(SOCS1).And investigate the diagnosis and prognosis prediction of SOCS1 in spinal TB patients.Method In dataset GSE83456,the x Cell scores of macrophages and macrophage subtypes was estimated.Peripheral blood monocytes from spinal TB and non-TB patients were collected for identification and comparison of macrophage subtypes using flow cytometry.Ferroptosis related differential expression(FRDEGs)were obtained from dataset GSE83456 and were verified using multiple GEO datasets.Further explore the role SOCS1 in diagnostic,therapeutic evaluation,drug resistance and bone destruction.QRT-PCR was used to verify the expression of FRDEGs in peripheral blood of patients with spinal TB.The relationship between the expression of FRDEGs and macrophage polarization was also analyzed.Result In dataset GSE83456,macrophages,M1-type macrophages,and M2-type macrophages were significantly elevated in the EPTB group.In clinical cases,M1 macrophages were significantly elevated in patients with spinal TB,and M1/M2 was higher in patients with spinal TB than in controls.In addition,SOCS1 was identified as a FRDEG and was verified in multiple GEO datasets.SOCS1 could work as a diagnostic factor in TB patients,and its expression significantly decreased after receiving standard anti-TB therapy.In dataset GSE144127,SOCS1 was significantly upregulated in osteoarticular TB.Furthermore,the expression of SOCS1 was higher in osteoarticular TB than other diseases that causing bone destruction.In addition,SOCS1 was positively correlated with the proportion of M1 macrophages.Conclusion The macrophages were M1 type polarization and the expression of SOCS1 is up-regulated after M1 macrophage polarization in spinal TB patients.In addition,the expression of SOCS1 in osteoarticular TB was significantly higher than ther diseases that causing bone destruction.Part 3 Changes of macrophage polarization and the SOCS1 expression after RAW264.7 cells stimulating by recombination ESAT-6Objective To investigate the changes of macrophage polarization and the SOCS1 expression after RAW264.7 cells stimulating by recombination ESAT-6 in vitro.Method TB infection model was established using RAW264.7 cells stimulating with recombination ESAT-6.The proportion of M1 and M2 macrophages after RAW264.7 macrophages stimulating by recombination ESAT-6 was detected using flow cytometry.Meanwhile,the expression changes of SOCS1 m RNA and protein in RAW264.7 stimulateing by recombination ESAT-6 were detected by q RT-PCR and western blot respectively.The changes of RAW264.7 stimulated by LPS,IL-4 and recombination ESAT-6 were compared.Result With the increase concentration of recombination ESAT-6,RAW264.7 macrophages tended to become M1 polarization.And when the concentration of recombination ESAT-6 increased to 8 μg/ m L,the M1 polarization tended to be stable.Compared with the lipopolysaccharide(LPS)and Interleukin 4(IL-4)stimulation,the polarization ratio after recombination ESAT-6 stimulation was similar to that of LPS stimulation.With the increase concentration of recombination ESAT-6 stimulation,SOCS1 expression was also increased gradually after M1 polarization.The expression of SOCS1 increased after ESAT-6 stimulation was similar to that of LPS stimulation.Conclusion Recombination ESAT-6 promotes M1 polarization and the expression of SOCS1 was up-regulated after M1 polarization of RAW264.7 macrophages.Part 4 Effect of SOCS1 expression on osteoclast differentiation of RAW264.7 cellsObjective To investigate the effect of SOCS1 on osteoclast differentiation of RAW264.7 cells.Method The key genes of osteoclast differentiation pathway regulated by SOCS1 were selected through bioinformatics analysis.The osteoclast differentiation model was established using RAW264.7 cells stimulating with macrophage colony-stimulating factor(M-CSF)+ receptor activator of nuclear factor kappa-B ligand(RANKL).Tartrate-resistant acid phasphate(TRAP)staining was used to observe osteoclast.The RAW264.7 cells were divided into four group including blank group,M-CSF + RANKL + NC group,M-CSF + RANKL group and M-CSF + RANKL + SOCS1-si RNA group.The expression of related genes and proteins was determined by q RT-PCR and western blot respectively after interventing with different factors.Result The expression levels of nuclear factor kappa b subunit 1(NF-κB1),nuclear factor kappa b subunit 2(NF-κB2)and nuclear factor of activated t cells 1(NFATC1)were increased after RAW264.7 cells were differentiated into osteoclasts.However,when SOCS1-si RNA was added in the stimulation process,the osteoclasts differentiation was interfered.TRAP staining showed that the number of osteoclasts were significantly decreased when SOCS1-si RNA was added in the stimulation process.Moreover,the m RNA and protein expression levels of NF-κB1,NF-κB2 and NFATC1 in M-CSF + RANKL group and M-CSF + RANKL + NC group were significantly higher than those in M-CSF + RANKL + SOCS1-si RNA group.Conclusion The expression of NF-κB1,NF-κB2 and NFATC1 was inhibited after using SOCS1-si RNA.SOCS1 promotes osteoclast differentiation by activating NF-κB1/NFATC1.Part 5 SOCS1 induces ferroptosis in patients with spinal TB leading to the destruction of disc tissueObjective The aim of this study was to investigate and analyze the relationship between SOCS1 and ferroptosis of intervertebral disc cells in patients with spinal TB.Method The intervertebral disc tissues of spinal TB patients and spinal trauma patients were collected for q RT-PCR and immunohistochemistry to estimate the expression of SOCS1 and glutathione peroxidase 4(GPX4).And then we analyze the relationship between SOCS1 and GPX4.Result QRT-PCR results showed that the expression of SOCS1 was significantly increased,while GPX4 was significantly decreased in the intervertebral disc tissues of patients with spinal TB.Similarly,immunohistochemical results indicated that the expression of SOCS1 was significantly increased and GPX4 gene was significantly decreased in the intervertebral disc tissue of patients with spinal TB.Conclusion SOCS1 may induce ferroptosis of intervertebral disc cells in patients with spinal TB by inhibiting the expression of GPX4.