Study On The Similarities And Differences Between Physiological Degeneration And Acupuncture Degeneration Of Intervertebral Disc In Rats

Part 1.Correlation between disc degeneration and ageObjective:To investigate the relationship between disc degeneration and age,and to provide a theoretical basis for the study of disc age-related degeneration.Methods:In this part,male SD rats at different months of age were divided into three groups with five rats in each group.3M group:3-month-old rats;Group 12M:12-month-old rats;24M group:24month-old rats.Disc degeneration was analyzed by X-ray,MRI,histopathology,immunohistochemistry and atomic mechanical microscopy in different groups.Results:The results showed that compared with the 3M group,the intervertebral spaces were clear and did not show significant osteophyte hyperplasia under the X-ray in the 12M group,the intervertebral spaces were slightly narrowed and there was osteophyte hyperplasia in the paraspinal bone in the 24M group,and DHI significantly decreased as the age increased.In group 12M,the signal of the visible disc on MRI T2 images was strong and homogeneous with a clear demarcation of nucleus pulposus and annulus fibrosus,while in group 24M,the visible disc on MRI T2 images still had some hyperintensity with water content and a clear demarcation of nucleus pulposus and annulus fibrosus,and the T2 signal intensity decreased significantly with increasing age.The histological score results also increased significantly with age in months.Immunohistochemical results showed that the expression levels of COL1,MMP3 and TNF-α increased significantly with the increase of age,while the expression levels of COL2 decreased significantly with the increase of age.AFM observations showed that the collagen fibrils of group 3M were well arranged,those of group 12M were more loosely arranged,and those of group 24m were scattered;The values of elastic modulus of fibrillar ring collagen fibrils,elastic modulus of nucleus pulposus collagen fibrils,and mean diameter of fibrillar ring collagen fibrils increased significantly with age,and the mean value of D-spacing did not differ among the three groups.Conclusions:Radiology,histopathology,related protein expression,and microstructure and mechanical characteristics indicated slow degeneration of the intervertebral disc with increasing age.Part 2.Correlation between disc degeneration and puncture injuryObjective:To investigate the relationship between disc degeneration and puncture injury,and to provide a theoretical basis for studying the early degeneration of acupuncture injury to the intervertebral disc.Methods:In this part,3-month-old SD rats were divided into three groups of five rats.Normal group(NG group):rats without pinprick manipulation;Acupuncture at degeneration for 2 weeks(acupuncture degeneration group-2 weeks,APDG2w group):rats underwent complete puncture(21g needle)of the caudal disc,2 weeks after puncture,to create the degeneration model;Acupuncture at degeneration for 4 weeks(acupuncture degeneration group-4 weeks,APDG-4w group):rats underwent complete puncture(21g needle)of the intervertebral discs,four weeks after puncture,to create the degeneration model.Disc degeneration was analyzed by X-ray,MRI,histopathological evaluation,immunohistochemistry,and atomic force microscopy in different groups.Results:In the APDG-2w group,some narrowing of the intervertebral space and no significant osteophyte hyperplasia were observed under X-ray;X-ray of APDG-4w group showed narrowing of the intervertebral space and osteophyte hyperplasia in the adjacent vertebrae.DHI in rats decreased significantly with longer puncture time.The APDG-2w group MRI T2 image visible nucleus pulposus structure appears as hypointense with narrowing of the intervertebral space;The APDG-4w group MRI T2 image showed that the nucleus pulposus structure was hypointense and the intervertebral space was narrowed.After puncture injury,T2 signal was significantly reduced in rats.On histological staining,in the APDG-2w group,the annulus fibrosus was disorganized and the nucleus pulposus was hypocellular.In the APDG-4w group,the annulus fibrosus was disorganized and the nucleus pulposus tissue was lost.The histopathological score significantly increased with longer puncture time.Immunohistochemistry showed that the amount of COL1,MMP3,and TNFα expression increased significantly with the time of puncture,and the amount of COL2 expression decreased significantly with the time of puncture.The AFM results showed that the NG group had well aligned collagen fibrils,the APDG-2w group had more loosely aligned collagen fibrils,the APDG-4w group had irregular distribution of collagen fibrils,and the values of elastic modulus of fibrillar ring collagen fibrils increased significantly with the extension of puncture time,and the average diameter of fibrillar ring collagen fibrils and the average D-spacing were not significantly different among the three groups.Conclusion:Acupuncture injury in rats causes damage to the structural integrity and mechanical environment of the intervertebral disc with increasing puncture time,from which imaging,histopathology,related protein expression as well as microstructural and mechanical features indicate rapid degeneration of the disc.Part 3.Similarities and differences between disc aging and puncture degenerationObjective:To investigate the similarities and differences between age and needle injury on disc degeneration.Methods:In this part,SD rats were divided into three groups of five rats each.Normal group(NG):3-month-old rats+2 weeks;Acupuncture degeneration group(APDG-2w):3-month-old rats underwent complete puncture(21g needle)of the intervertebral disc 2 weeks after puncture to create a degeneration model;Physiological degeneration group(PDG):aged rats were normally housed for 24 months.Disc degeneration was analyzed by X-ray,MRI,histopathological evaluation,immunohistochemistry,and atomic force microscopy in different groups.Results:The narrowing of intervertebral spaces was observed under X-ray in APDG-2w group and PDG group.Disc DHI was significantly lower in the APDG-2w and PDG groups and even lower in the APDG-2w group.The MRI T2 image of the visible nucleus pulposus structure in the APDG-2w group showed hypointensity,and the MRI T2 image of the PDG group still showed partial hyperintensity of the visible disc.T2 signal intensity of the intervertebral disc decreased in both the APDG-2w and PDG groups,with a greater decrease in the APDG-2w group.Histological scores increased in both the APDG-2w and PDG groups,with higher scores in the APDG-2w group.Immunohistochemistry showed that the amount of COL1,MMP3,and TNF-α expression rose significantly in the APDG-2w and PDG groups,with a more pronounced rise in the PDG group.The amount of COL2 expression was significantly lower in the APDG-2w and PDG groups,and the decrease was more pronounced in the PDG group.AFM observations showed that the NG group had well aligned collagen fibrils,the APDG-2w group had more loosely aligned collagen fibrils,and the PDG group had scattered collagen fibrils.The elastic modulus of collagen fibrils was significantly increased in the APDG-2w and PDG groups,and was significantly higher in the APDG-2w than in the PDG group.The mean diameter of collagen fibrils in the PDG group was significantly increased compared with the NG and APDG-2w groups,and the mean D-spacing did not differ among the three groups.Conclusion:From the comparison of imaging,histopathological,microstructural and mechanical characteristics,degeneration was more severe in the acupuncture group than in the aging group,but the immunohistochemical indexes and the mean diameter of collagen fibers were higher in the aging group than in the acupuncture group.Part 4.Proteomic similarities and differences between aging intervertebral disc and acupuncture degeneration intervertebral discObjective:To investigate proteomic differences in age and acupuncture injury induced disc degeneration.Methods:In this part,SD rats were divided into three groups of three rats each.Normal group(NG):3-month-old rats+2 weeks;Acupuncture degeneration group(apdg-2w):3-month-old rats underwent complete puncture(21g needle)of the intervertebral disc 2 weeks after puncture to create a rapid degeneration model;Physiological degeneration group(PDG):rats aged for 24 months were normally housed.After successful modeling,the rat disc samples were collected,the disc samples were pretreated,proteins were extracted and concentrations were determined,the proteome was examined using the label free method,and the data results were subjected to bioinformatics analysis.The Acan,Mapk3,Comp and Myoc were verified by PRM method and Western blot.Results:The results showed a total of 2109 identified proteins in the sample,and 1317 proteins completed the quantification.The number of co-expressed proteins in NG,APDG-2w and PDG groups was 698.Compared with the APDG-2w and PDG groups,100 proteins were up-regulated and 82 were down regulated.Differential proteins for subcellular localization showed a maximum of 225 differential proteins in the cytoplasm,followed by the nucleus,for 196 differential proteins.Differential proteins performing functional domain analysis showed the highest proportion of lipoprotein/cytosolic fatty acid binding protein family differential proteins.Go analysis of differential proteins showed that the most differential proteins in cell component related proteins,and differential protein KEGG pathway analysis showed that the proportion of differential proteins in the complement system and Staphylococcus aureus infection pathway was higher.The results of PRM protein validation showed that the Acan,Mapk3,Comp and Myoc proteins were significantly different between the three groups.There was a correlation between the development of acupuncture disc degeneration and downregulation of Acan,Comp,and Myoc proteins,a correlation with upregulation of Mapk3 proteins.There was a correlation between aging disc degeneration and downregulation of Acan proteins,a correlation with upregulation of Mapk3、Comp and Myoc proteins.Conclusions:There is differential expression of protein in degenerative discs with aging and acupuncture.