| Purpose:In this paper,the effects of deep needling "Huantiao" on the functional recovery of sciatic nerve and the regeneration of nerve myelin sheath in rats with sciatic nerve injury(SNI)were observed by means of behavioral test,electrophysiological test and immunofluorescence staining,to explore the key time node of nerve myelin regeneration,clarify the time course that deep needling"Huantiao" can promote nerve myelin regeneration,and analyze the mechanism of deep needling "Huantiao" to promote SNI nerve myelin regeneration from the hippo signal pathway(HIPPO)signal pathway.Material and method:Paper 1 Study of the optimal intervention time of deep stimulation of "Huantiao point" in rats with sciatic nerve injury1 animal grouping and intervention81 male rats with SPF grade SD were randomly divided into model group,acupuncture group and positive drug group,with 28 rats in each group.All rats were used to prepare SNI rat model by crush injury method.The rats were routinely raised for 28d after modeling in the model group and were not otherwise disposed.The following day of modeling in the acupuncture group,rats were deeply punctured at the right ring hop points,and the depth of the needle entry was approximately 12-17mm subcutaneously(acupuncture needle tip touched the sciatic nerve trunk).Tremor maneuvers were performed 3-5 times a day by pinprick once the needle touched the nerve trunk,and the needle was left in place for 20 min for 28 consecutive days(stop for 1 day every 6 consecutive days of treatment).The day after modeling in the positive drug group,2mg/mL ganglioside GM1 solution at 10 mg/mL was injected into the tail vein once daily for 28d(stop for 1 day every 6 consecutive days of treatment).2 extraction and index detectionIn each group,three rats were randomly selected on 0d,4d,8d,12d,16d,20d,24d and 28d after successful modeling and before successful modeling,and the sciatic nerve functional index(SFI)was measured using a self-made rat footprinting walking box,and the motor nerve conduction velocity(MCV)of the sciatic nerve was measured using a BL-420 biomechanical test instrument.After that,they were killed by cervical amputation,the right sciatic nerve tissue was removed,and prepared paraffin sections for HE staining to observe the pathological changes in rat sciatic nerves,and immunofluorescence staining to observe the content of myelin sheath of sciatic nerve and S-100 β,GAP-43 expression levels.Paper 2 Study on the Mechanism of Deep Acupuncture "Huantiao Point" on Myelin Regeneration in SNI Rats Based on HIPPO Pathway1 animal grouping and intervention120 male rats with SPF grade SD were randomly divided into sham,model,acupuncture,and positive drug groups,with 30 rats in each group.Rats in the model group,acupuncture group and positive drug group were used to produce SNI rat model by crush injury method.Sham group was given sham operation and routine husbandry,no other disposition.They were routinely raised for 16 d after modeling in the model group and were not otherwise disposed.The following day of modeling in the acupuncture group,rats were deeply punctured at the right ring hop points,and the depth of the needle entry was approximately 12-17mm subcutaneously(acupuncture needle tip touched the sciatic nerve trunk).Tremor maneuvers were performed 3-5 times a day by pinprick once the needle touched the nerve trunk,and the needle was left in place for 20min for 16 consecutive days.The following day of modeling in the positive drug group,2mg/mL ganglioside GM1 at 10 mg/mL was injected into the tail vein once daily for 16 d.2 extraction and index detectionAt the end of the intervention,two rats in each group were randomly selected for the extraction and identification of scdes,and the remaining rats were examined by SFI using a self-made rat footprinting walking box,sural muscle EMG using a multifunctional electromyograph,and sciatic nerve MCV and sensory nerve conduction velocity(SCV)using a BL-420 biomechanical test.After that,the rats were sacrificed,the sciatic nerve tissues were collected,and paraffin sections were prepared from 1/2 of the nerve tissue for immunohistochemical staining to visualize CDH1 protein expression in sciatic nerve;The remaining 1/2 nerve tissues were frozen at-80℃ and used for Western blot analysis of protein expression of SAV1,Mst1/2,Lats1/2,Mob1,CTGF,Gli2,AREG,FGF1,as well as Real-time PCR for YAP mRNA and TAZ mRNA expression in sciatic nerves.Paper 3 Study on the mechanism by which deep spines " "Huantiao acupoint ""mediates HIPPO signaling by intervening Schwann cell exosome pathway to promote nerve regeneration in a rat model of sciatic nerve injury.1 animal grouping and intervention40 male rats with SPF grade SD were randomly divided into model exosome group,deep pricking Huantiao exosome group,and 20 rats in each group were treated with crush injury to prepare SNI rat model.The rats retained in paper 2 were taken and subjected to SCDEs extraction and identification.In the model exosome group,SCDEs from the model group in paper Ⅱ were given tail vein injections after modeling,and 3 μ L/mouse,every 3d for a total of 6 injections.The day after modeling began in the deep pricking Huantiao exosome group,SCDEs from the acupuncture group in paper Ⅱ were given tail vein injections,3 μL/mouse,every 3d for a total of 6 injections.2 extraction and index detectionAt the end of the intervention,all rats were examined for SFI,sural muscle EMG,sciatic nerve MCV and SCV,and the expressions of CDH1,SAV1,Mst1/2,Lats1/2,Mob1,CTGF,Gli2,AREG,FGF1protein,YAP mRNA and TAZ mRNA in sciatic nerves.The specific operation is the same as that in paper 2.Results:Paper 1 Study of the neurorestorative processes of deep stimulation of " Huantiao point " in rats with sciatic nerve injury1.Effects of deep acupuncture " Huantiao acupoint " at different times on SFI in SNI rats.Compared with before modeling,the levels of SFI of rats at 0d of intervention in each group were significantly decreased(P<0.05).Compared with intervention day 0d,the levels of SFI in the positive drug group and acupuncture group gradually increased from the 8d of intervention,and the levels of SFI in the model group gradually increased from the 12d of intervention(P<0.05).There were no statistical differences in the levels of SFI among rats in the model group,positive drug group and acupuncture group on the 0d and 4d of intervention(P>0.05).Intervention at 8 d,12 d,16d,20d,24d and 28d,compared with the model group,the levels of SFI of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).2.Effects of deep acupuncture " Huantiao acupoint " at different times on MCV in SNI rats.Compared with before modeling,the levels of MCV of rats at 0d of intervention in each group were significantly decreased(P<0.05).Compared with intervention day 0d,the levels of MCV in the positive drug group and acupuncture group gradually increased from the 8d of intervention,and the levels of MCV in the model group gradually increased from the 12d of intervention(P<0.05).There were no statistical differences in the levels of MCV among rats in the model group,positive drug group and acupuncture group on the 0d and 4d of intervention(P>0.05).Intervention at 8 d,12 d,16d,20d,24d and 28d,compared with the model group,the levels of MCV of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).3.Effects of deep stimulation of " Huantiao acupoint " at different times on the pathological morphology of SNI ratsAt 0 d and 4 d of intervention,the sciatic nerves of each group had obvious local fragmentation,sparse distribution and arrangement of nerve fibers,disintegration and loss of myelin,interstitial edema,and occasional proliferation of individual SCs.On the 8 d and 12 d D of intervention,there was no significant improvement of sciatic nerve in model group compared with before;The nerve fibers in the positive drug group began repair;Nerve fiber repair was obvious in acupuncture group.On the 16th and 20 d of intervention,a small number of SCs proliferated in the model group;A large number of SCSs in the positive drug group proliferated,and there was de novo myelination;A large number of proliferated SCs in the acupuncture group were arranged in an orderly fashion with axon formation.On the 24 d and 28 d of intervention,newly generated nerve fibers were seen in the model group;A large number of regenerated nerve fibers were seen in the positive drug group and acupuncture group,which were surrounded by thicker myelin sheaths,axons were clear and nearly normal in diameter,nerve fibers were arranged in relatively tight bundles,and the extent of nerve repair regeneration in the acupuncture group was higher than that in the positive drug group.4.Effects of deep stimulation of " Huantiao point " at different times on the speed of nerve regeneration in SNI ratsOn the 0 d and 4d of intervention,the myelin content of sciatic nerve in each group was significantly decreased compared with that before modeling.On the 8 d and 12 d days of intervention,the myelin content of sciatic nerve in the model group showed no significant improvement compared with before;An increase in myelin content began to occur in the acupuncture group and the positive drug group,in which the increase was more pronounced in the acupuncture group.At 16 d,20 d,24 d and 28 d of intervention,the myelin content of sciatic nerve in the model group was a small increase,and the myelin content of acupuncture group and positive drug group was significantly higher than that in the model group,and gradually and substantially increased,and the myelin content of acupuncture group was higher than that of positive drug group.5.Effect of deep stimulation of " Huantiao point " at different times on the expression levels of S-100β and GAP-43 of sciatic nerve in SNI ratsCompared with before modeling,the expression levels of S-100β in rats at 0d of intervention in each group were significantly decreased(P<0.05).Compared with intervention day 0d,the expression levels of S-100β and GAP-43 of sciatic nerve in the positive drug group and acupuncture group gradually increased from the 4d of intervention,and the expression levels of S-100β and GAP-43 of sciatic nerve in the model group gradually increased from the 8d of intervention(P<0.05).There were no statistical differences in the expression levels of S-100β and GAP-43 of sciatic nerve among rats in the model group,positive drug group and acupuncture group on the 0d and 4d of intervention(P>0.05).Intervention at 4d,8d,12d,16d,20d,24d and 28d,compared with the model group,the expression levels of S-100β and GAP-43 of sciatic nerve of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).Paper 2 Experimental study on deep stimulation of " Huantiao acupoint " affecting remyelination via HIPPO signaling in a rat model of sciatic nerve injury1.Behavioral test resultsSFI:Compared with the sham group,the levels of SFI in the model rats were significantly decreased(P<0.05).Compared with the model group,the SFI levels of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).Lower limb muscle strength:Compared with the sham group,the lower limb muscle strength in the model rats were significantly decreased(P<0.05).Compared with the model group,the lower limb muscle strength of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).2.Results of nerve electrophysiology testingGastrocnemius electromyography:Compared with the sham group,the action unit potential amplitude and wave width in the gastrocnemius muscle EMG in the model rats were significantly decreased(P<0.05).Compared with the model group,the action unit potential amplitude and wave width in the gastrocnemius muscle EMG of rats in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).Nerve conduction velocity:Compared with the sham group,the MCV and SCV of sciatic nerve in the model rats were significantly decreased(P<0.05).Compared with the model group,the MCV and SCV of sciatic nerve in the acupuncture group and the positive drug group were significantly higher,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).3.Molecular biological test results(1)Effects of deep acupuncture " Huantiao acupoint " on the expression levels of SAV1,Mst1/2,Lats1/2,Mob1,CTGF,Gli2,AREG,FGF1 protein in SNI ratsCompared with the sham group,the expression levels ofSAV1,Mst1/2,Lats 1/2,Mob 1 protein in the model rats were significantly increased(P<0.05).Compared with the model group,the expression levels of SAV1,Mst1/2,Lats1/2,Mob1 protein of rats in the acupuncture group and the positive drug group were significantly decreased,and the expression levels of the above indicators in the acupuncture group were significantly lower than those in the positive drug group(P<0.05).Compared with the sham group,the expression levels of CTGF,Gli2,AREG,FGF1 protein in the model rats were significantly decreased(P<0.05).Compared with the model group,the expression levels of CTGF,Gli2,AREG,FGF1 protein of rats in the acupuncture group and the positive drug group were significantly increased,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).(2)Effects of deep acupuncture " Huantiao acupoint " on the expression of CDH1 in SNI ratsCompared with the sham group,the relative expression of CDH1 in the sciatic nerve in the model rats were significantly decreased(P<0.05).Compared with the model group,the relative expression of CDH1 in the sciatic nerve of rats in the acupuncture group and the positive drug group were significantly increased,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).(3)Effects of deep acupuncture " Huantiao acupoint " on the expression of YAP mRNA,TAZ mRNA in SNI ratsCompared with the sham group,the expression levels of YAP mRNA,TAZ mRNA in the sciatic nerve in the model rats were significantly decreased(P<0.05).Compared with the model group,the expression levels of YAP mRNA,TAZ mRNA in the sciatic nerve of rats in the acupuncture group and the positive drug group were significantly increased,and the expression levels of the above indicators in the acupuncture group were significantly higher than those in the positive drug group(P<0.05).Paper 3 Study on the mechanism by which deep spines " Huantiao acupoint "mediates HIPPO signaling by intervening Schwann cell exosome pathway to promote remyelination in a rat model of sciatic nerve injury.1.SCDEs identification resultsWhen the isolated scdes were imaged by TEM at 80 kV,the structure of the vesicles was typical of a tea cup like shape,with double membrane wrapping and low electron density in the interior,whereas the electron density in the edge part was high,which was compatible with the basic morphology of exosomes.Nanoparticle tracking analysis(NTA)was used to measure the size of the vesicles,and the results showed that most of the vesicles were distributed between 60 and 180 nm with a mean value of 138 nm,which is consistent with the diameter of exosomes.Western blot analysis showed positive expression of exosome specific marker proteins such as CD9 and CD63 in vesicles,which is consistent with the characteristics of exosomes.The above experimental results can confirm that the vesicles isolated in this work are exosomes.2.Behavioral test resultsSFI:Compared with the model exosome group,the SFI of rats in the deep pricking Huantiao exosome group was significantly higher(P<0.05).Lower limb muscle strength:Compared with the model exosome group,the lower limb muscle strength of rats in thedeep pricking Huantiao exosome group was significantly increased(P<0.05).3.Results of nerve electrophysiology testingGastrocnemius electromyography:Compared with the model exosome group,the action unit potential amplitude and wave width in the gastrocnemius muscle EMG of rats in the deep pricking Huantiao exosome group were significantly increased(P<0.05).Nerve conduction velocity:Compared with the model exosome group,the MCV and SCV of the sciatic nerve of rats in the deep pricking Huantiao exosome group were significantly higher(P<0.05).4.Molecular biological test results(1)Effects of SCDEs on the expression levels of SAV1,Mst1/2,Lats1/2,Mob1,CTGF,Gli2,AREG,FGF1 protein in SNI ratsCompared with the model exosome group,the protein expression levels of SAV 1,Mst1/2,Lats 1/2,Mob 1 in the sciatic nerve of the deep pricking Huantiao exosome group were all significantly decreased(P<0.05).Compared with the model exosome group,the protein expression levels of CTGF,Gli2,AREG,FGF1 in the sciatic nerve of the deep pricking Huantiao exosome group were significantly increased(P<0.05).(2)Effects of SCDEs on the expression of CDH1 in SNI ratsCompared with the model exosome group,the relative expression of CDH1 in sciatic nerve of the deep pricking Huantiao exosome group was significantly higher(P<0.05).(3)Effects of SCDEs on the expression of YAP mRNA,TAZ mRNA in SNI ratsCompared with the model exosome group,the expression levels of YAP mRNA and TAZ mRNA in the sciatic nerve of the deep pricking Huantiao exosome group were significantly increased(P<0.05).Conclusion:1.Deep needling of "Huantiao point" can improve the specific marker of SCs of injured nerves in SNI rats S-100 β from the 4th day and the expression level of nerve growth associated protein GAP-43.The content of myelin sheath began to increase on the 8th day,and increased with the increase of intervention time,reaching the peak after 16 days.2.Deep stimulation of " Huantiao points " can obviously improve the limb motor function and electrophysiological function of injured nerves in SNI rats,accelerate the sciatic nerve functional recovery and nerve regeneration,and prevent the denervation atrophy of gastrocnemius muscle.3.Deep stimulation at " Huantiao acupoint " can up regulate CDH1 and YAP mRNA,TAZ mRNA and CTGF,Gli2,AREG and FGF1 expression and down regulate SAV1,Mst1/2,Lats1/2 and Mobl expression in the injured sciatic nerve of SNI rats,indicating that deep stimulation at " Huantiao acupoint " may promote remyelination and accelerate the repair of injured nerve by regulating HIPPO signaling pathway.4.SNI model rats after being subjected to the intervention of deep stimulation of "Huantiao acupoint ",the extracted SCDEs of sciatic nerve can play the same role as deep stimulation of " Huantiao acupoint ",which is speculated that deep stimulation of "" Huantiao acupoint "" may regulate HIPPO signaling pathway by regulating Schwann cell-derived exosomes pathway,and then improve remyelination in injured sciatic nerve.In conclusion,the scientific hypothesis that deep stimulation of " Huantiao acupoint " affected HIPPO signaling pathway in the sciatic nerve injured organism through early intervention with Schwann cell-derived exosomes pathway,and then intervened in the regeneration of myelin of sciatic nerve,and promoted the repair of sciatic nerve injury is theoretically feasible. |
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