| Background Catalpol is one of the major effective constituent of Rehmannia glutinosa Libosch.The previous study shown Catalpol promoted axon outgrowth of the primary cortex neuron in vitro,and enhanced axon sprouting or dendrite growth, improved axon sprouting or enhanced synaptic reorganization in vivo. As yet it is not known whether Catalpol play vital role in the restorations of the peripheral nerves injury.Based on the high incidence of sciatic nerve injury as a kind of representative`s peripheral nerves injury, sciatic nerve injury has less effective treatment. To investigate the effects and mechanism of Catalpol on sciatic nerve injury will be benefit to clinical treatment, which has very important scientific value and clinical significance.Objective of this study is as follows:1 To observe the effects of Catalpol on the behavioristics in the mice after nerve crush injury.2 To observe the effects of Catalpol on neuron in spinal cord and gastrocnemius muscle functional recovery after sciatic nerve crush injury.3 To confirm the role of m-TOR signaling pathway on the neural regeneration induced by Catalpol after sciatic nerve crush injury.Part I Catalpol improved neuroethology functional recovery in mice after sciatic nerve crush injuryObjective:To examine neuroethology functional recovery treatment with Catalpol in mice after sciatic nerve crush injury.Methods1 To establish animal models of mice with crushed sciatic nerves, and administered intraperitoneally daily for 7d with Catalpol(10mg/kg body weight), equal volume normal saline were used in model and sham group, respectively.2 Effect of catalpol on pain tolerance threshold and Walking track analysis was measured at 1,4,7 days post-surgery.The sciatic function index(SFI) was calculated for the assessment of motor nerve recovery.3 The gastrocnemius muscles from both the operated and contralateral, un-operated limbs of mice were removed, and the muscle wet weight ratio was calculated between injured and contralateral muscle. And the fixed muscle specimens were stained with Haematoxylin and Eosin(HE) staining. The cross-sectional area(CSA) of the muscle fibers was measured.4 In order to study the effect of Catalpol in the recovery of blood-nerve barrier after sciatic nerve injury and observe the permeability difference of blood nerve barrier between the three groups by intravenous injection of Evans Blue at 7 days after sciatic nerve crush injury.Results1. Catalpol improved neuroethology functional recovery(1) Catalpol raised the pain tolerance threshold: Compared to model(12.13s±2.82,12.91s± 4.15), Catalpol(27.06 s ±2.17, 34.48s±3.72) significantly raised the pain tolerance threshold at 4,7d after sciatic nerve crush injury, respectively(p<0.05, p<0.01).(2) Catalpol improved the movement function recovery: Compared to model(-91.88±2.63、-88.32±4.19), SFI scores were significantly increased in catalpol(-82.52±2.12、-70.12±1.90)treatment group(p<0.05)at 4, 7 d after sciatic nerve crush injury, respectively.2. Catalpol prevented the denervated skeletal muscle atrophy induced by sciatic nerve crush injury.(1) the muscle wet weight ratio,defined as the ratio of muscle wet weight of the operated side to the contralateral non-operated side,is a more reliable measure assay than the muscle wet weight itself.The results of muscle wet weight ratio showed that the wet weight ratio of Catalpol(0.825±0.039 vs 0.586±0.046, p< 0.05) groups was significantly higher than that of model group;(2) Compared to model group, Catalpol obviously increased cross-sectional area of denervated skeletal muscle fiber.(774.67μm2± 65.07 vs 452.10μm2± 50.41, p <0.05).(3) The morphologic of gastrocnemius: The sham group gastrocnemius maintained circular and polygonal muscle fibers, polynuclear and nuclear located below the surrounding muscle basement membrane. The model group resulted in significant injury as demonstrated by gastrocnemius muscle cells edema, muscle fiber atrophy were appear distinctly angularity, as well as appear vacuolization. However,catalpol treatment ameliorated severe gastrocnemius muscle damage, muscle cells not only arranged in neat rows and closely but also sarcolemma nuclei increased.4. Catalpol attenuated blood nerve barrier leakage associated with sciatic nerve crush injury: Compared to model group, less Evans Blue into the endoneurium was observed in Catalpol group on the 7d after crush, the result was further confirmed by the quantification of Evans Blue.Conclusion Catalpol could enhanced the functional recovery and and protects the blood-nerve barrier and attenuates Evans Blue leakage after sciatic nerve injury.PartⅡ The impact of Catalpol on motor neuron unit( spinal cord-sciatic nerve-muscle)Objective: To observe the effects of Catalpol on the structure morphologic changes of motor neuron unit, including skeletal muscle,motor end plate and enzyme activity of Ach E,and study Catalpol protection of spinal cord anterior horn neurons after sciatic nerve injury of mice.Methods1. To establishanimal models of mice with crushed sciatic nerves,and the mice were randomly divided into five groups(n=8 in each group), and administered intraperitoneally daily with Catalpol(10mg/kg), Rapa(0.5mg/kg), Rapa+Cat(0.5mg/kg+10mg/kg) and equal volume normal saline were used in model and sham group, respectively. These agent treatments lasted for 7days。2. To examine the neurons loss using Nissl staining in spinal cord and the apoptosis status of spinal anterior horn cells using TUNEL dyeing, to detect Bax, Bcl-2 protein level in spinal crod neuron using Western blot.3. Mean while, to observe the regenerated sciatic nerve fiber and measured the myelin thickness by transmission electron microscope.4. To observe the activity of Motor End Plate(MEP) with acetylcholinesterase enzyme staining in gastrocnemius muscles.Whole-mount gold chloride staining was used too bserve the Catalpol effects on extent of recovery of nerve endings both of motor and sensory.Results1. Catalpol inhibited apoptosis of neurons in spinal cord(L4-L6)(1) Nissl staining shown that: 7 days after operation, the number of the Nissl body was reduced obviously, a large number of Nissl bodies was dissolves and cell necleus displacement can be seen. After catalpol treatment,neuron swelling was attenuated, the number of the Nissl body was increased significantly and bigger α neurons, longer dendrite of the neurons were observed.(2) TUNEL staining shown that: Compared with model(54.28±10.71),Catalpol(16.39±2.64) significantly reduced the number of TUNEL dyeing positive neurons(P<0.05).Bax protein level in model group is strikingly higher than sham group(P<0.05),While Catalpol reduced Bax protein expression. At the same time Catalpol up-regulated Bcl-2protein level obviously. The ratio of the Bcl-2 and Bax was increased significantly after treatment with catalpol.2. Catalpol improved destroyed myelin recovery(1) Sciatic nerve axons are wrapped by thinner layers of myelin after crush injury,and Catalpol reversed this phenomemon, the thicker layers of myelin was observed.Rapa aggravated myelin injury, much debris and demyelinate phenomemon were observed. Catalpol reversed Rapa effects on myelin change.(2) Because of swelling, the thickness of the myelin of the sciatic nerve fibers increased obviously after injury when compared to the sham group(0.1147μm±0.0336)( P <0.05); Compared to the model group(0.2310μm±0.0414), the thickness of the myelin of the sciatic nerve fibers decreased in Catalpol group(0.1846μm±0.0245) dramaticly(P<0.05),and Rapa seriously destroyed structure of the myelin of the sciatic nerve fibers and the thickness of the myelin was not been calculated and Catalpol reversed Rapa effects on the myelin of the sciatic nerve fibers.3. Catalpol improved MEPs degenerationafter sciatic nerve crush injury.(1)Catalpol increased intact MEPs number after sciatic nerve crush injuryAfter sciatic nerve crush injury, it was observed in model and Rapa group that MEPs were light stained with a blurred margin, burr-like morphology and an irregular shape.Catalpol treatment enhanced ACh E staining with a more complete shape that was nearly to the sham group; ACh E staining became deeper, suggesting an increase in enzyme activity after treatment with Catalpol.The number of intact MEPs in model(5.7±2.19) and rapa group(5.1±1.37) reduced obviously(P<0.05). Catalpol(12.8±3.06) treatment increased the number of intact MEPs significantly( P < 0.05), and Catalpol reversed Rapa effects on MEPs degeneration after sciatic nerve crush injury, including the shape and the number of intact MEPs in muscle.(2) Catalpol improved MEPs degeneration by Whole-mount gold chloride stainingThe model group showed the nerve endings had a claw-like appearance. The Catalpol group showed the axonal tree-like formation and intertwined cylindrical endings of sensory nerve endings. The Rapa group nerve endings showed a pink staining and had a filamentous-like appearance and Catalpol reversed Rapa effects on MEPs degeneration after sciatic nerve crush injury.Conclusion:Catalpol could delay the degeneration of motor end plate and increase the activities of the acetylcholinase on the MEP,that promote the restore of peripheral nerve after the injuries. Catalpol maybe participated in anti-apoptosis creaction process of neurons after sciatic nerve injury, and it has relative to m-TOR signaling pathway.Part Ⅲ The effects of actived m-TOR signaling pathway on the neural regeneration by Catalpol after sciatic nerve injuryObjective:To study Catalpol’s effects is relative to m-TOR signaling pathway or not on the neural regeneration by Catalpol after sciatic nerve injury.Methods1. The methods to establish the model and the group divided were as same as to Part II.2. Effect of catalpol on pain tolerance threshold and Walking track analysis was measured at 1,4,7 days post-surgery.The sciatic function index(SFI) was calculated for the assessment of motor nerve recovery.3. The protein of Akt, p-Akt, m-TOR, p-m-TOR, P70S6 K, p-P70S6 K, PTEN and p-PTEN expressions was detected by Western blot analyses in all groups after different administration following surgery.4. GAP-43 expression of the relevant segment spinal cord and sciatic nerve after sciatic nerve injury was observed and detected by immunostaining and Western blot,respectively.Results1. Catalpol reversed Rapa effects on neuroethology functional recovery(1) Catalpol raised the pain tolerance threshold: Compared to Rapa(12.70s± 3.79,14.17 s ± 3.05), catalpol(27.06 s ± 2.17, 34.48 ± 3.72) and Catalpol combine Rapa group(32.51 s ± 1.88, 27.06s± 4.24) significantly raised the pain tolerance threshold at4,7d after sciatic nerve crush injury, respectively(P <0.05, P < 0.01).(2) Catalpol improved the movement function recovery:Compared to Rapa(-89.28 ±3.47).SFI scores were significantly increased in Catalpol(-82.52±2.12) treatment group(p<0.05)at 4d after sciatic nerve crush injury, respectively.While, Catalpol combine Rapamycin compared with Rapamycin group were no significant differenceat 4d after sciatic nerve crush injury(P > 0.05).Compared to Rapa group(-88.32 ± 4.19), SFI scores were significantly increased in Catalpol(-70.12 ± 1.90) and Cat + Rapa group(-81.42 ± 2.16),( p<0.05) at 7d after sciatic nerve crush injury.2. Catalpol was actived m-TOR signaling pathway and inhibited the PTEN expression(1) Western blot result shown that: compared to the model group, Catalpol significantly up-regulated the p-m-TOR and p-P70S6 K protein level(P<0.05). While Rapa inhibited p-m-TOR and p-P70S6 Kprotein expression. At the same time Rapa was also down-regulated p-Akt protein level obviously, and Catalpol reversed Rapa effects on m-TOR signaling pathway relative protein. As for PTEN, model and Rapa group significantly up-regulated PTEN expression compared to the sham group(P<0.05), and Catalpol decreased PTEN expression(P<0.05 vs model) and reversed Rapa effects on PTEN(P<0.05 vs Rapa).2. Catalpol improved GAP-43 and BDNF expressionImmunostaining staining and Western blot shown that: GAP-43 positive stained mainly occured in the spinal cord anterior horn cells and sciatic nerve,the expression of GAP-43 in the spinal cord and sciatic nerve was increased obviously after treatment with Catalpol.And the number of positive expression of GAP-43 in Catalpol group(26±4.6) were significantly higher than that of model group(9±1.2, P < 0.05); Rapa decreased the number of positive expression of GAP-43, and Catalpol reversed Rapa decreased effects on GAP-43 expression. As for BDNF expression, Catalpol up-regulated expression of BDNF, and Rapa down-regulated BDNF expression, Catalpol reversed Rapa decreased effects on BDNF expression.Conclusion: Catalpol promoted and improved nerve regeneration via the m-TOR signaling pathway activation after sciatic nerve injury in mice. |
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