| [Objective]Study the effect of Tuina therapy on axon growth guidance mechanism and signal transduction, to further explore Tuina mechanism of promoting recovery of peripheral nerve injury and improve the theoretical basis of Tuina in the treatment of peripheral nerve injury, for the clinical application of Tuina in the treatment of peripheral nerve injury, improve symptoms and prognosis of patients to provide strong scientific evidence.[Methods]The massage manipulation simulator simulates Tuina manipulation, and intervenes the sciatic nerve (sciatic nerve injury, SNI) model rats by quantitative and qualitative treatment. Study the effect of Tuina therapy on axon growth guidance mechanism through the following three aspects:1 By incline board test and heat pain tolerance threshold to observe the behavior condition of rats. Using transmission electron microscope to observe and analysis sciatic nerve axon, myelin and Schwann cells ultrastructure changes, so as to further explore the morphological evidence of Tuina therapy promoting the recovery of peripheral nerve injury.2 Based on ultrastructural evidence of Tuina in the treatment of peripheral nerve injury, using immunohistochemistry and molecular biology methods, to detect the axon outgrowth guidance factor and its receptor of spinal cord and sciatic nerve, interpret the influence of Tuina on axon guidance mechanisms.3 To further validate Tuina is able to regulate axonal growth guidance, the Rho GTPase inhibitor NSC23766 blocking axonal growth oriented mechanism of Rho GTPase signaling transduction, observe the changes of rat sciatic nerve function and changes of neuronal cytoskeleton protein F-actin and Tublin. Analysis Tuina is through the regulation of axon growth and guidance mechanism in signal transduction to regulate growth cone cytoskeleton, to improve the regeneration of nerve growth and get the accurate orientation, so as to restore the function of target organs.[Results]1 Tuina can promote the recovery of nerve ultrastructure in SNI model rats1.1 The behavioral results:After modeling 7d, compared with the model group the animal behavior performance of Tuina group is the same (P> 0.05). After 20 times of Tuina treatment, the inclined plate test and thermal resistance threshold results of Tuina and model group were significantly different (P< 0.05), and close to the level of sham operation group (P< 0.05). This indicates that after Tuina treatment, the motor and sensory function of rats have been a certain degree of recovery.1.2 Electron microscope results:under electron microscope, after modeling 28d model group myelin was damaged, myelin structure fuzzy, loose, vacuolar degeneration, axonal atrophy or disappearance, Schwann cell mitochondria vacuolated, cells become necrosis, that indicates nerve damage is serious, and is consistent with the behavior performance of rats. After 20 times of Tuina treatment, treatment group rat sciatic nerve was observed under electron microscope myelin stratification, vacuolation, no obvious axonal swelling, mitochondria swollen, cristae and vacuolization, part of Schwann cell vacuolization or mitochondrial edema, the extent of damage is lighter than the model group. Compared with the model group, the thickness of myelin sheath and axon diameter of myelinated nerve of Tuina group were significantly increased (P< 0.05), and gradually close to the level of normal group (P< 0.05).These results suggest that Tuina can promote the repair and regeneration of peripheral nerve ultrastructure, and provide ultrastructural morphological evidence for the recovery of nerve injury in SNI rats.2 Tuina can promote the expression of axon growth guidance factor and its receptor in SNI model rats2.1 Expression changes of Netrinl and mRNAAfter intervention 0 times, average integral optical density, the relative content of protein and mRNA expression of Netrinl in L3-L5 segments of the spinal cord and sciatic nerve, compared with normal group, the model group, model control group and Tuina group are significantly increased (P< 0.05), sham operation group and the normal group are no difference (P> 0.05). Intervention 20 times, the model group, model control group and Tuina group were significantly higher than that in normal group (P< 0.05), model group and model control group had no significant difference (P> 0.05), Tuina group increased significantly (P< 0.05) compared with model group and model control group.2.2 Expression changes of DCCIntervention 0 times, model group, model control group and Tuina group of spinal cord and nerve in DCC the average integral optical density were significantly increased compared with the normal group (P< 0.05), sham operation group and the normal group had no difference (P> 0.05). After 20 times of intervention, in spinal cord, model group, model control group and Tuina group were significantly higher than the normal group (P< 0.05), there was no significant difference between the model group and model control group (P> 0.05), Tuina group compared with model group and model control group were significantly increased (P< 0.05); and in the nerve, model group, model control group and the normal group have no difference (P> 0.05), but the Tuina group is still significantly higher than the normal group (P< 0.05), there are also significant differences compared with the model control group and model group (P< 0.05).2.3 Expression changes of UNC5AIntervention 0 times, average integral optical density of UNC5A in the spinal cord and nerve of model group, model control group and Tuina group were significantly increased compared with the normal group (P< 0.01), sham operation group and the normal group had no difference (P> 0.05). Intervention after 20 times of model group in the spinal cord, model control group and Tuina group were significantly higher than the normal group (P< 0.05), there was no significant difference between the model group and model control group (P> 0.05), Tuina group compared with model group and model control group were significantly increased (P< 0.05); and in nerve, model group, model control group and Tuina group were significantly higher than the normal group (P< 0.01), there was no significant difference between the model group and model control group (P> 0.05), Tuina group compared with model group and model control group were significantly increased (P< 0.01).2.4 Expression changes of Slit2 and mRNAAfter intervention 0 times, average integral optical density, the relative content of protein and mRNA expression of Slit2 in L3-L5 segments of the spinal cord and sciatic nerve, compared with normal group, the model group, model control group and Tuina group are significantly increased (P< 0.05), sham operation group and the normal group are no difference (P> 0.05). Intervention 20 times, the model group, model control group and Tuina group were significantly higher than that in normal group (P< 0.05), model group and model control group had no significant difference (P> 0.05), Tuina group increased significantly (P< 0.05) compared with model group and model control group.2.5 Expression changes of RobolAfter intervention 0 times, average integral optical density of Robol in spinal cord and nerve of the model group, model control group and Tuina group were significantly increased (P < 0.05) compared with normal group, sham operation group and the normal group were no difference (P> 0.05). After Intervention 20 times, in spinal cord the model group, model control group and Tuina group were significantly higher than that in normal group (P< 0.01), but the three groups had no significant difference (P> 0.05), and in nerve model group and model control group were significantly higher than that in normal group (P< 0.05), and the two groups had no significant difference (P> 0.05), Tuina group is very significant (P< 0.01) compared with normal group, and compared with model group and model control group also have significantly increased (P< 0.05).These results suggest:Tuina can promote the synthesis and secretion of Netrinl and its receptor DCC/UNC5A in nerve and corresponding spinal cord segments; Tuina can promote the expression of Slit2 in nerve and corresponding spinal cord segments, but only can promote the expression of Robo1 in the nerve; Tuina can maintain the expression equilibrium between DCC and UNC5A, Netrinl and Slit2.3 Tuina can affect axon guidance through Rho GTPase signal transduction3.1 Sciatic function index (SFI) results:after modeling 1 d and 7 d, SFI results of model, Tuina, Tuina+inhibitors and Tuina+saline group compared with the sham operation group were significantly lower (P< 0.01). After modeling 14 d, four groups still were significantly lower than control group (P< 0.01), but the Tuina and Tuina+saline group compared with the 7 d have a rise. After modeling 21 d, model group and Tuina+inhibitor group were significantly lower than control group (P< 0.01);Tuina and Tuina+saline group compared with model group had significantly higher (P<0.05), and with the normal group there was no significant difference (P>0.05);Tuina+inhibitor group higher than model group (P<0.05), but significantly lower than the Tuina and Tuina+saline group (P< 0.05). After modeling 28d, four groups compared with 21 d have a certain degree of recovery;Model group and the Tuina+inhibitors have a little rise, but still lower than control group (P< 0.05);Tuina and Tuina+saline group compared with model group had significantly higher (P< 0.05), and have been up to the level of normal group (P> 0.05);Tuina+inhibitor groups higher than model group (P< 0.05), but still lower than Tuina and Tuina+saline group (P< 0.05).3.2 Results of cytoskeletal proteins:intervention 0 times, the average integral optical density of F-actin and Tublin in model group, Tuina group, Tuina+inhibitor group and Tuina +saline group were significantly decreased compared with the sham operation group (P< 0.05). Intervention after 20 times, the four group were significantly lower than the sham operation group (P< 0.05), but the Tuina group and Tuina+saline group compared with the model group increased significantly (P< 0.05), Tuina+inhibitor group compared with model group, there was no difference (P> 0.05), and significantly lower than the Tuina group and Tuina+saline group (P< 0.05). The results of immunofluorescence is the same as immunohistochemistry:in sham operation group, F-actin and Tublin showed immunoreactive particles, a green fluorescent color high brightness, color depth, fluorescence intensity was significantly higher than other groups; F-actin and Tublin of model group were less green fluorescence positive granules, when intervention 20 times, the fluorescence intensity significantly weaker than in the other groups; Fluorescence intensity and the color distribution of Tuina group and Tuina+saline group can not be distinguished the difference by naked eye, but the intervention 20 times, two groups’ color depth enhance obvious compared with model group in the fluorescence intensity; 20 times after the intervention, the Tuina+inhibitor group colored particles less than Tuina group and Tuina+saline group, but fluorescence intensity is still higher than model group.These results suggest:after inhibition of axon guidance signal transduction of Rho GTPase, the promoting effect of Tuina on cytoskeleton protein F-actin and microtubule protein Tublin, and the sciatic nerve function are affected.[Conclusion]1 Tuina can promote the regeneration of the SNI rat sciatic nerve myelin, axon recovery, reducing edema of the Schwann cell cytoplasm and mitochondria, have a significant role in promoting peripheral nerve repair and regeneration of the ultrastructure. This provides morphological evidence for Tuina treating with peripheral nerve injury.2 Tuina can promote axon guidance factor Netrinl and its receptor DCC/UNC5A, Slit2 and its receptor Robo1 expression, and can maintain their attraction and repulsion signal balance, this may be mechanism of Tuina can promote axonal accurate orientation, and successfully control target organ.3 Tuina can promote expression of cytoskeleton protein F-actin and Tublin by axon guidance signal transduction, regulating reconstruction and motion of axonal cytoskeletal structure, thereby affecting the growth of axon orientation and nerve regeneration.Above conclusions confirming each other, indicate that Tuina can regulate regeneration axon growth and guidance through the axon guidance mechanisms, for the recovery of neural function and morphology, and finally the target organ get correct reinnervation, so as to promote the peripheral nerve injury symptoms recovery.
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