| Objective:1.Based on the FAK-PI3K-AKT signaling pathway,to explore the effect mechanism of"discerning structure regulating balance Tuian" in the treatment of cervical spondylosis.2.To verify the scientific hypothesis that"discerning structure regulating balance Tuian" can inhibit the apoptosis of chondrocytes in intervertebral disc and relieve neck pain through the integrin β1-mediated FAK-PI3K-AKT signaling pathway,thus achieving the treatment of cervical spondylosis.3.To reveal the effective mechanism of"discerning structure regulating balance Tuian"in the treatment of cervical spondylosis,and to provide experimental basis for clinical Tuian therapy of cervical spondylosis.Methods:1 To observe the clinical effect of "discerning structure regulating balance Tuian"in the treatment of cervical spondylosisA total of 62 patients with cervical spondylosis from the Bone Injury Department of Shenzhen Baoan Chun Traditional Chinese Medicine Treatment Hospital and the Tuian department of the Third Affiliated Hospital of Changchun University of Traditional Chinese Medicine were included.32 patients in the treatment group were treated with "discerning structure regulating balance Tuian",and 30 patients in the control group were treated with medium-frequency pulse electric therapy.Visual analogue scale(VAS),cervical joint motion measurement(ROM score),cervical dysfunction index(NDI score)and X-ray cervical physiological curvature of the two groups were compared before and after treatment,so as to evaluate the curative effect before and after treatment.2 It is confirmed that"discerning structure regulating balance Tuian"can inhibit the apoptosis of chondrocytes in intervertebral disc through the integrin β1-mediated FAK-PI3K-AKT signaling pathway,relieve neck pain,and reach the scientific hypothesis of treating cervical spondylosisNew Zealand rabbits were subjected to cervical spondylosis modeling for 8 weeks and intervention for 2 weeks with the lower head position method.After the modeling,rabbits in the blank group,rabbits in the modeling group and rabbits after the intervention were taken neck X-ray and CT images to observe the physiological curvature of the cervical vertebra,vertebral space and vertebral arrangement.Cervical intervertebral disc tissues before and after the intervention were taken,and the intervertebral disc HE staining and Masson staining were performed to observe the changes of the fibrous annulus and its structure(C3/4,C4/5,C5/6).Intervertebral discs(C3/4,C4/5,C5/6)of rabbits in blank group,model rabbits and rabbits after intervention were collected.After grinding and centrifugation,supernatate was taken to detect the changes of inflammatory factors TNF-α and IL-1β in intervertebral discs,and the changes of inflammatory factors in rabbits in each group were evaluated.HE staining was performed on the neck muscle tissue of rabbits before and after the intervention,and the arrangement of muscle fibers and inflammatory cell infiltration were observed.TUNEL method was used to detect cell apoptosis in intervertebral disc tissues(C4/5,C5/6).Immunohistochemical and Western blot analysis were performed to detect the expression changes of apoptotic proteins Bax,Bcl-2 and caspase-3 in intervertebral disc tissues before and after intervention.The expression levels of pathway-related proteins integrin β1,p-FAK,p-PI3K and p-AKT were detected.Results:1 Clinical study on the treatment of cervical spondylosis by "discerning structure regulating balance Tuian"based on the academic thought of differentiation and treatment1.1 Comparison of VAS score:Through the analysis of independent sample t test,patients in both groups were significantly lower after treatment than before treatment,and the difference was statistically significant(P<0.05).The pain improvement effect of the treatment group was better than that of the control group.1.2 Comparison of ROM scores:By comparing the changes of anterior flexion,backward flexion,left flexion,right flexion and left and right rotation Angle before and after treatment,patients in both groups were reduced after treatment compared with before treatment,and the difference was statistically significant(P<0.05).It can be seen that the treatment group improved the cervical motion of patients better than the control group.1.3 Comparison of NDI score:Through rank sum test analysis,both groups of patients were lower after treatment than before treatment,and the difference was statistically significant(P<0.05).It can be seen that the treatment group is better than the control group in improving neck symptoms and dysfunction.1.4 Comparison of physiological curvature of X-ray cervical vertebrae:Through the analysis of rank sum test,there were significant differences in the treatment group compared with before treatment(P<0.05),but no significant differences in the control group compared with before treatment(P>0.05).It can be seen that the treatment group improved the physiological curvature of patients’ cervical vertebrae better than the control group.2 The influence of "discerning structure regulating balance Tuian"on the cervical structure and cervical disc of static injury type rabbit modelCompared with the blank group,the cervical curvature of rabbits in the model group was significantly changed by lateral cervical radiographs combined with CT,and even cervical spine "inverting arch" appeared,with uneven vertebral edges,mild bone hyperplasia,irregular arrangement,and unequal vertebral space.The cervical curvature of rabbits in the latter group was straightened,and the vertebral body arrangement was more orderly than that in the model group.After comparison,the cervical curvature of the rabbits in the group was improved,the phenomenon of "antiarch" of the cervical spine basically disappeared,and the vertebral body was arranged more neatly.The cervical lateral film of rabbits in the blank group showed that the physiological curvature was normal,the vertebral edges were flat and neatly arranged,and the cervical space was basically equal.The histomorphologic changes of the annulus fibrosus of intervertebral disc in rabbits in each group were as follows:The annulus fibrosus of rabbits in the blank group showed obvious hierarchical and neatly arranged structure with uniform cell distribution;Compared with the blank group,the fiber annulus of the model group showed loose and disordered fiber arrangement,more tearing,swelling and degeneration of cells.The fibrous ring arrangement of the two groups was relatively neat,the level was relatively clear,and the cell morphology was slightly improved.3 The influence of "discerning structure regulating balance Tuian"on the homeostasis of intervertebral disc in rabbit model with static injuryAfter grinding and centrifugation,the supernatant was taken to test the changes of inflammatory factors TNF-α and IL-1β in the intervertebral discs,and the inflammatory factors in the discs of the Diagnosis and Balancing Tuian group and the Posterior group were both reduced compared with those of the model group,but the effect of the Diagnosis and Balancing Tuian group was better.The results of Masson staining showed that the structure of healthy intervertebral discs remained intact and their collagen was neatly arranged.In this case,the annulus fibrosus was blue,while the nucleus pulposus and its cytoplasm were red or reddish-brown,with a clear boundary between the two.In contrast,the medulla in the model group showed severe fibrosis,with more pronounced fragmentation of the nuclei,deepening of the boundary between the fibrous ring and the medulla,severe breakage of the fibrous ring,and darker staining.After treatment,the discs of the discs in the Diagnostic and Balancing Tuian and Posterior Group showed improvement in collagen content and the gap between the annulus fibrosus and the nucleus pulposus,and the expression of type II collagen,proteoglycans,and matrix metalloproteinase(MMP-13)in the discs was better than that of the model group by Western blotting,which demonstrated the efficacy and restoration of internal environmental homeostasis of the manipulation.and the restoration of homeostasis in the internal environment.4 The effect of "discerning structure regulating balance Tuian" on apoptosis of intervertebral disc chondrocytes in static injury type rabbit modelThe number of apoptotic cell nuclei in the counting field of view was measured using an image analysis programme.The percentage of apoptotic cells in the total number of cells was calculated at 400 times the field of view,i.e.the apoptotic index.Compared with the model group,the apoptotic cell death rate in the cervical intervertebral discs of rabbits in both the Tui Na group and the posterior group decreased significantly(P<0.01),but the data of the Tui Na group were slightly better than those of the posterior group(P<0.05).This indicated that the acupressure techniques had an inhibitory effect on apoptotic cells.The expression of apoptotic proteins Bcl-2,Bax and Caspase-3 in the cervical intervertebral discs of rabbits in each group was detected by immunohistochemistry and Western Blot.The results of Western Blot can be corroborated by the results of immunohistochemistry,which indicated that the manipulation techniques in the Diagnosis and Balance Tui Na group and the Hou Qun group could increase the positive expression of Bcl-2 and Bax in the intervertebral discs,and at the same time inhibit the expression of Caspase-3.Caspase-3 expression in the intervertebral disc to inhibit chondrocyte apoptosis.However,compared with the latter group,the effect of the Diagnostic and Balancing Tui Na group was more significant.5 Based on the FAK-PI3K-AKT signaling pathway,the influence of "discerning structure regulating balance Tuian"on the static injury type rabbit model was studiedAfter the experiment,the intervertebral disc tissues of rabbits in each group were analyzed by Western Blot to detect the protein expression levels of p-FAK,p-PI3K and p-AKT.After treatment,compared with the model group,the relative expression levels of integrin-β1/β-actin,P-FAK/β-actin,P-PI3K/β-actin and P-Akt/β-actin in the Tiaohe Tuian group and the post-group were all increased,but there was statistical significance in the Tiaohe Tuian group(P<0.01).There was no statistical significance in the latter group(P>0.05).Conclusion:1.The neck balancing method guided by the academic thought of "discerning structure regulating balance Tuian" has shown significant clinical efficacy in improving pain symptoms of patients,so it is worth promoting in clinical practice;2."discerning structure regulating balance Tuian"has a better regulating effect on the instability of cervical vertebra structure and the changes of intervertebral disc fibroring in static injury type rabbit model than the treatment of a single post-group Tuian;3."discerning structure regulating balance Tuian"may restore the dynamic balance of neck muscle,relieve pain,and inhibit the apoptosis of chondrocytes in the endplate of cartilaginous intervertebral disc through the mechanical signaling pathway FAK-PI3K-AKT.This helps to restore the environmental homeostasis and elasticity of the intervertebral disc,and then restore the static balance of the cervical spine,slow down the process of intervertebral disc degeneration,and rebuild the mechanical balance of the cervical spine. |
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