Based On The JNK Signal Pathway To Explore The Mechanism Of Acupuncture On Nerve Repair In Rats With Sciatic Nerve Injury

Purpose:The therapeutic mechanism of acupuncture on sciatic nerve injury was studied on the whole,cell and molecular level.To explore the effect and mechanism of acupuncture on the repair of sciatic nerve injury in rats,we observed the expression of neuromotor function and tissue structure,serum and oxidative damage markers,JNK signal pathway mediated apoptosis related proteins and Schwann cell specific markers.At the same time,by observing the changes of cell cycle and apoptosis of rsc96 cell line and the expression of apoptosis related protein mediated by JNK signal pathway,the protective effect and mechanism of acupuncture serum effectors on oxidative damage of neural Schwann cells were explored.Material and method1 In vivo animal experiment（Papers 1 to 3）1.1 animal grouping and modeling70 SPF grade SD rats were divided into sham operation group（SHAM）,model group（SNCI）,superficial needling group（SEA）,deep needling group（DEA）and non acupoint deep needling group（NEA）.In addition to SHAM group,the other four groups used classical clamp method to prepare the rat sciatic nerve crush injury model.1.2 intervention methodsAfter 1 day of model making,each group began acupuncture treatment according to the corresponding intervention measures.The "Huantiao" point（GB30）on the same side of model making was selected as the treatment site in the two groups of SEA and DEA.The acupuncture depth in the SEA group was about 0.5cm,which was appropriate to touch the muscle tissue under the acupuncture site;the acupuncture depth in the DEA group was about 1.5cm,which was appropriate to touch the nerve trunk.In NEA group,the line between "Huantiao" point（GB30）on the same side of the model and the lateral edge of the knee joint,about 1cm from the distal end of the injury site,was selected as the treatment site,and the appropriate depth of penetration was about 1.5cm to reach the nerve trunk.Each group was slightly inserted and twirled for 3 times,then connected with the positive pole of the electroacupuncture instrument,and the tail connected with the negative pole.The frequency was 2.0Hz,the current was 2.0m A,once a day,20 mins each time,for 14 days.1.3 sampling and index testThe motor function of sciatic nerve was evaluated in all rats before taking samples,then the electrophysiological test was performed under anesthesia,and the blood from abdominal aorta was used to detect the levels of SOD,MDA and 8-OHd G in serum by ELISA.The sciatic nerve tissue and dorsal root ganglion tissue were taken from the rats,and one of them was fixed in 2.5%glutaraldehyde,which was used to observe the ultrastructure of the tissues under transmission electron microscope;three of them were fixed in 4% paraformaldehyde,which was used to observe S100 protein expression by immunohistochemistry,p-JNK,p-c-jun,Bcl-2,Bax,cleaved-caspase-3,cleave d-caspase-8 protein expression;the remaining tissues cryopreserved at-80 ℃ were used to detect SOD,MDA,8-OHd G levels in the nerve by ELISA;JNK,c-jun,Bcl-2,Bax,Caspase-8,caspase-3 m RNA expression was detected by RT-PCR;p-ERK,p-p38,p-JNK,TNF-α,p-c-jun,Bcl-2,Bax,cleaved caspase-3,cleaved caspase-8,S100 protein expression was detected by Western blot.2 in vitro cell experiments（Paper 4）2.1 preparation of acupuncture serum and cell grouping15 SD rats were divided into blank group and treatment group.There were 10 rats in blank group and 5 rats in treatment group.The modeling method and intervention method of blank group were the same as those of sham group in vivo.The modeling method and intervention method of treatment group were the same as those of deep stab group in vivo.They were treated for 14 days continuously.Blood was taken from abdominal aorta 0.5h after the last treatment,and the serum was inactivated and filtered to prepare acupuncture serum.According to the different conditions of induction and intervention,the cells were divided into three groups: Control group（Control）,Model group（H₂O-₂）and Acupunture serum group（H₂O-₂ +acupunture serum）.2.2 cell culture and screening of intervention conditionsRSC96 cell line was resuscitated and cultured to 3 generations.CCK-8 method was used to screen the conditions of H₂O-₂-induced apoptosis and acupuncture serum intervention cells.2.3 index detectionFlow cytometry and annexin V / PI double staining were used to detect cell cycle and apoptosis;ELISA was used to detect TNF-α expression in supernatant;immunofluorescence was used todetect S100 and cleared caspase-3 protein expression;after adding SP600125,Western blot was used to detect p-JNK,p-c-jun,Bcl-2,Bax,cleaved Caspase-8 and cleaved caspase-3 protein expression.Results（1）Animal experiments in vivo Paper 1 An experimental study on the effect of acupuncture at "Huantiao " point（GB30）on the function,morphological repair and oxidative damage of sciatic nerve in rats1.Evaluation of motor function of sciatic nerve in experimental ratsThe sciatic nerve function index（SFI）of rats with crush injury decreased significantly（P <0.01）.After treatment,compared with SNCI group,the SFI of SEA and DEA group increased significantly（P < 0.01）,and the increase degree of DEA group was higher than that of SEA group（P< 0.01）.2.Electrophysiological detection of sciatic nerve in experimental ratsThe motor nerve conduction velocity（MNCV）decreased significantly（P < 0.01）.After treatment,compared with SNCI group,MNCV of SEA and DEA group increased significantly（P <0.01）,and the increase degree of DEA group was higher than that of SEA group（P < 0.05）.3.Ultrastructural observation of sciatic nerve in experimental ratsIn SHAM group,the myelin sheath and Schwann cells were intact.In SNCI group,the myelin sheath was deformed,the intact Schwann cells disappeared,and a small amount of unmyelinated nerve fibers appeared.In the SEA group,the myelin structure was restored,but the arrangement was loose,there was lamellar separation;in the DEA group,the myelin structure was relatively complete,and the myelinated nerve fibers with uneven thickness and a large number of Schwann cells were seen to proliferate.In NEA group,the recovery of myelin sheath structure was poor,almost no Schwann cells were found.4.Ultrastructural observation of dorsal root ganglion in experimental ratsIn each group,there was no obvious abnormality in the ultrastructure of DRG,the myelin sheath was basically complete,and Schwann cells with complete structure could be seen at the edge of myelin sheath.5.The effect of acupuncture at "Huantiao" point（GB30）on the markers of oxidative damage in serum of ratsCompared with sham group,SOD level of snci group decreased significantly（P < 0.01）.Compared with SNCI group,SOD level in SEA and DEA group was significantly higher（P < 0.01）,and SOD level in DEA group was higher than that in SEA group（P < 0.01）.Compared with SHAM group,the levels of MDA and 8-OHd G in SNCI group were significantly higher（P < 0.01）.Compared with SNCI group,MDA and 8-OHd G levels in SEA and DEA group were significantly lower（P < 0.01）,MDA and 8-OHd G levels in DEA group were lower than those in SEA group,MDA and 8-OHd G levels in NEA group were lower than those in SNCI group but much higher than those in SEA and DEA group（P < 0.01 or P < 0.05）.6.Effect of acupuncture at "Huantiao" point（GB30）on oxidative damage markers of sciatic nerve in ratsCompared with SHAM group,SOD level of SNCI group decreased significantly（P < 0.01）.Compared with SNCI group,SOD level in SEA and DEA group was significantly higher（P < 0.01）,and SOD level in DEA group was higher than that in SEA group（P < 0.01）.Compared with SHAM group,the levels of MDA and 8-OHd G in SNCI group were significantly higher（P < 0.01）.Compared with SNCI group,MDA and 8-OHd G levels in SEA and DEA group decreased significantly（P < 0.01）,MDA and 8-OHd G levels in DEA group were lower than those in SEA group（P < 0.01）,MDA levels in NEA group were lower than those in SNCI group but much higher than those in SEA and DEA group（P < 0.01）,8-OHd G levels in NEA group were lower than those in SNCI group（P < 0.01）,but higher than those in DEA group（P < 0.01）Paper 2 Experimental study on JNK signal pathway of sciatic nerve injury model rats by three acupuncture points "Huantiao" point（GB30）1.Effect of acupuncture at "Huantiao" point（GB30）on MAPK signal pathway of sciatic nerve and dorsal root ganglion in ratsThe expression of p-ERK protein in sciatic nerve tissue in DEA group was lower than that in other four groups（P < 0.05）.Compared with SHAM group,the expression of p-p38 protein in SNCI,SEA and NEA group increased（P < 0.05）,while that in DEA group decreased（P < 0.05）,and that in NEA group increased（P < 0.05）.Compared with SHAM group,the expression of p-JNK protein in other four groups increased（P < 0.05）;compared with SNCI group,the expression of DEA group decreased（P < 0.05）.Compared with SHAM group,the expression of p-ERK protein in DRG increased in DEA andNEA group（P < 0.05）.Compared with DEA group,NEA group protein expression increased（P <0.05）.Compared with SHAM group,the expression of p-p38 protein in other four groups increased（P < 0.05）;compared with SNCI group,the expression of SEA group decreased（P < 0.05）;compared with SEA and DEA group,the expression of NEA group increased（P < 0.05）.Compared with SHAM group,the expression of p-JNK protein in SNCI,SEA and NEA group increased（P < 0.05）;compared with SNCI and SEA group,the expression of DEA group decreased（P < 0.05）and NEA group increased（P < 0.05）.2.Effect of acupuncture at "Huantiao" point（GB30）on JNK signal pathway of sciatic nerve and dorsal root ganglion on apoptosis in rats2.1 RT-PCR was used to detect the expression of JNK,c-jun,Bcl-2,Bax,Caspase-8 and caspase-3m RNA in rat sciatic nerve and dorsal root ganglionCompared with SHAM group,the expression of JNK1 m RNA in sciatic nerve increased significantly in SNCI,SEA and NEA group（P < 0.05）,and decreased significantly in DEA group（P< 0.05）.There was no significant difference in the expression of JNK2 m RNA in each group（P >0.05）.Compared with SHAM group,the expression of JNK3 m RNA in the other four groups increased significantly（P < 0.05）,and there was no significant difference between the two groups（P > 0.05）.Compared with SHAM group,the expression of c-jun m RNA in other four groups was significantly higher（P < 0.05）;compared with SNCI group,the expression of DEA group was significantly lower（P < 0.05）,and there was no significant difference among SNCI,SEA and NEA（P > 0.05）.Compared with SHAM group,the expression of Bcl-2 / Bax m RNA in SNCI,SEA and NEA group decreased significantly（P < 0.05）;compared with SNCI group,the expression in DEA group increased significantly（P < 0.05）;the expression in NEA group was significantly lower than that in SNCI group（P < 0.05）.There was no significant difference in caspase-3 m RNA expression among the groups（P > 0.05）.Compared with the SHAM group,the expression of Caspase-8 m RNA in the other four groups was significantly increased（P < 0.05）;compared with the SNCI group,the expression in the other three groups was significantly reduced（P < 0.05）,in which there was no significant difference between SEA and NEA（P > 0.05）,and the expression in the DEA group was lower than that in the NEA group（P < 0.05）.Compared with SHAM group,the expression of JNK1 m RNA in DRG was significantly higher in SNCI group（P < 0.05）,and lower in SEA and DEA group（P < 0.05）.Compared with SHAMgroup,the expression of JNK2 m RNA in SNCI group was significantly higher（P < 0.05）;compared with SNCI group,the expression in the other three groups was significantly lower.Compared with SHAM group,the expression of JNK3 m RNA in SNCI,SEA and DEA group increased significantly（P < 0.05）;compared with SNCI group,the expression of SEA,DEA and NEA group decreased significantly（P < 0.05）;compared with SEA and DEA group,the expression of NEA group decreased significantly（P < 0.05）.Compared with SHAM group,the expression of c-jun m RNA in SNCI and SEA group increased significantly（P < 0.05）;compared with SNCI group,the expression of SEA,DEA and NEA group decreased significantly（P < 0.05）;there was no significant difference between SEA and NEA group（P > 0.05）,and the expression of DEA group was lower than the two groups（P < 0.05）.Compared with SHAM group,the expression of Bcl-2 / Bax m RNA in SNCI and SEA group decreased significantly（P < 0.05）;compared with SNCI group,the expression in SEA and DEA group increased significantly（P < 0.05）;the expression in DEA group was higher than that in SEA group and NEA group（P < 0.05）.Compared with SHAM group,the expression of caspase-3m RNA in other four groups increased significantly（P < 0.05）;compared with SNCI group,the expression of SEA and DEA group decreased significantly（P < 0.05）,and the expression of DEA group was lower than NEA group（P < 0.05）.Compared with SHAM group,the expression of caspase-8 m RNA in SNCI,SEA and NEA group was significantly higher（P < 0.05）;compared with SNCI group,the expression in DEA group was significantly lower（P < 0.05）,but there was no significant difference between SEA and NEA group（P > 0.05）.2.2 the effect of acupuncture at "Huantiao" point（GB30）on the expression of p-JNK,p-c-jun,Bcl-2,Bax,cleaved-caspase-3 and cleaved-caspase-8 in the sciatic nerve and dorsal root ganglion of ratsIn the sciatic nerve tissue,the fluorescence intensity of p-JNK protein was lower in each group,and there was no significant difference between the groups.The fluorescence intensity of p-c-jun protein was lower in each group.Compared with SHAM group,the intensity of SNCI and NEA group increased significantly;compared with SNCI group,the intensity of SEA and DEA group decreased significantly,while NEA group slightly decreased.The fluorescence intensity of Bcl-2protein was lower in each group,and there was no significant difference between the groups.Compared with SHAM group,the intensity of Bax protein in SNCI,SEA and NEA group increased significantly,and compared with SNCI group,the intensity of DEA group decreased significantly.The fluorescence intensity of cleaved-caspase-3 protein was significantly different in each group,theintensity of SHAM group and DEA group was lower,the intensity of SEA and NEA group was higher,and the intensity of SNCI group was the highest.The fluorescence intensity of cleaved-caspase-8 protein was significantly different in each group.The intensity of SNCI group was very high,and the other four groups were low.In the DRG,the fluorescence intensity of p-JNK protein was lower in each group,and there was no significant difference between the groups.The fluorescence intensity of p-c-jun protein was lower in each group,higher in SNCI group,and lower in the other four groups.The fluorescence intensity of Bcl-2 protein was lower in each group,and there was no significant difference between the groups.Bax protein fluorescence intensity was higher in each group.Compared with SHAM group,SNCI and NEA group increased significantly;compared with SNCI group,SEA and DEA group decreased significantly.The fluorescence intensity of cleaved-caspase-3 protein was significantly different in each group,the intensity of SHAM group and DEA group was lower,the intensity of SEA and NEA group was higher,and the intensity of SNCI group was the highest.The fluorescence intensity of cleaved-caspase-8 protein was lower in each group,and there was no significant difference between the groups.2.3 Western blot method was used to detect the effect of acupuncture at Huantiao on the expression of TNF-α,p-c-jun,Bcl-2,Bax,cleared caspase-3 and cleared caspase-8 in the sciatic nerve and dorsal root ganglion of ratsThe expression of TNF-α protein in sciatic nerve was lower in SHAM group,higher in SNCI group（P < 0.05）,and lower in DEA group than in SNCI group（P < 0.05）.Compared with SHAM group,the ratio of Bcl-2 / Bax protein in SNCI group decreased significantly（P < 0.05）,SEA and DEA increased significantly（P < 0.05）,and DEA group was superior to SEA group（P < 0.05）.There was no significant difference in the expression of p-c-jun protein in SHAM,SNCI,SEA and NEA groups（P > 0.05）.Compared with SHAM group,SNCI and NEA group increased significantly（P <0.05）,SEA and DEA decreased significantly（P < 0.05）,DEA group was better than SEA group（P <0.05）.Compared with SHAM group,the protein of cleaved caspase-8 increased significantly in SNCI group（P < 0.05）,and decreased significantly in SEA and DEA group（P < 0.05）.The expression of TNF-α protein in DRG was lower in SHAM group,higher in SNCI group（P< 0.05）,and lower in DEA group than in SNCI group（P < 0.05）.Compared with SHAM group,the ratio of Bcl-2 / Bax protein in SNCI group decreased significantly（P < 0.05）,SEA and DEAincreased significantly（P < 0.05）,and DEA group was superior to SEA group（P < 0.05）.Compared with SHAM group,p-c-jun protein in SNCI group increased significantly（P < 0.05）,DEA decreased significantly（P < 0.05）.Compared with SHAM group,the protein of cleaved caspase-3 in SNCI group increased significantly（P < 0.05）,SEA and DEA decreased significantly（P < 0.05）,and there was no significant difference between the two groups（P > 0.05）.There was no significant difference in the expression of cleaved caspase-8 protein in SHAM,SNCI,SEA and NEA groups（P > 0.05）,but in DEA group（P < 0.05）.Paper 3 The effect of the fourth acupuncture at "Huantiao" point（GB30）on S100 protein of Schwann cells in rats with sciatic nerve injury1.Effect of acupuncture on S100 protein expression in Schwann cells of sciatic nerve in ratsCompared with SHAM group,S100 expression decreased in SNCI group（P < 0.05）.Compared with SNCI group,the expression of SEA and DEA group increased（P < 0.05）,and DEA group was higher than SEA group（P < 0.05）.（2）In vitro cell experiment Paper 4 The experimental study on the effect of JNK signal pathway regulated by acupuncture serum on the apoptosis of RSC96 cells induced by H₂O-₂1.Effect of acupuncture serum on the activity of RSC96 cells induced by H₂O-₂After 6h of H₂O-₂ intervention,the cell activity of 0-50 μ M group decreased with the increase of H₂O-₂ concentration;the cell activity of 50-200 μ M group did not change significantly;the cell activity of 200-800 μ M group increased with the increase of H₂O-₂ concentration.After 6 hours of intervention with 50 μ m H₂O-₂,the inhibition rate of cell proliferation was relatively high,and the inhibition rate was about 80%.Therefore,the final concentration of H₂O-₂ was 50 μ mol / L,and the intervention time was 6 hours as the experimental conditions for H₂O-₂ induced rsc96 cell injury.After 24,48 and 72 hours,the cell viability decreased with the increase of serum concentration.The difference of cell viability of RSC96 cells incubated with 10% acupuncture serum at different time was small,and the inhibition rate of cell proliferation was low,16%,22% and 14%,respectively.Therefore,10% acupuncture serum was selected as the experimental condition of RSC96 cells incubated with acupuncture serum.The cell viability of the control group was higher than that of the control group after 24,48 and72 hours.The cell viability of H₂O-₂ group was low,and the cell survival rate was about 23%,20%and 18% respectively after 24,48 and 72 hours of culture.The cell viability of H₂O-₂ + acupunture serum group was significantly different,and the cell survival rate was about 40%,84% and 66%respectively after 24,48 and 72 hours of culture.Therefore,48 h of acupuncture serum intervention time was chosen as the experimental condition for the incubation of RSC96 cells with acupuncture serum,at this time,the cell survival rate increased most significantly.2.Effect of acupuncture serum on cell cycle of RSC96 induced by H₂O-₂Compared with the control group（go / G1 phase: 37.87%;S phase: 41.13%;G2 / M phase:13.60%）,go / G1 phase decreased to 25.50%（P < 0.05）and S phase increased to 50.87%（P < 0.05）.Compared with the cells of H₂O-₂ group,the S-phase decreased to 32.33%（P < 0.05）and the G2 / M phase increased to 18.30%（P < 0.05）in H₂O-₂ + acupunture serum group,which indicated that H₂O-₂ could block the cell cycle in S-phase,and acupuncture serum could effectively relieve the S-phase block caused by H₂O-₂.3.Effect of acupuncture serum on apoptosis of RSC96 cells induced by H₂O-₂In the control group,the majority of normal cells were in the control group,and the number of early apoptotic cells and late apoptotic cells were less.Compared with the control group,the number of apoptotic cells in the early stage and the late stage increased significantly in the H₂O-₂ group（P <0.05）.Compared with the H₂O-₂ group,the early and late apoptotic cells in the H₂O-₂ + acupunture serum group were significantly reduced（P < 0.05）,but still more than those in the control group（P <0.05）.4.The effect of acupuncture serum on the level of TNF-α in the supernatant of RSC96 cellsThe results showed that there was no significant change in TNF-α level in the control group（P > 0.05）.The level of TNF-α in cell supernatant was increased by H₂O-₂（P < 0.05）,and reached the peak at 24 hours.Acupunture serum began to decrease at 24 hours and decreased at 36 hours（P <0.05）.5.Effect of acupuncture serum on JNK signal pathway and apoptosis of RSC96 cells5.1 detection of S100 and cleaved caspase-3 protein expression in RSC96 cells with acupuncture serum by immunofluorescenceS100 protein fluorescence intensity was higher in each group,and there was no significant difference between the groups.The fluorescence intensity of cleaved caspase-3 protein,control group and H₂O-₂ + acupunture serum group were lower than that of H₂O-₂ group.5.2 Western blot analysis of the effect of acupuncture serum on the expression of p-JNK,p-c-jun,Bcl-2,Bax,cleaved caspase-8,cleaved caspase-3 protein in RSC96 cellsCompared with the control group,the expression of p-JNK protein in H₂O-₂ group was significantly increased（P < 0.05）,and decreased after adding SP600125（P < 0.05）.Compared with the control group,the expression of p-c-jun protein in H₂O-₂ group increased significantly（P < 0.05）,and decreased after adding SP600125（P < 0.05）.Compared with the control group,the Bcl-2 / Bax protein ratio in the H₂O-₂ group decreased significantly（P < 0.05）;compared with the H₂O-₂ group,the H₂O-₂ + acupunture serum group increased significantly（P < 0.05）;after adding SP600125,it increased significantly（P < 0.05）.Compared with the control group,the expression of cleaved caspase-3 protein in H₂O-₂ group increased significantly（P < 0.05）;compared with H₂O-₂ group,the expression of H₂O-₂ + acupunture serum and H₂O-₂ + acupunture serum + SP600125 group decreased（P < 0.05）.Compared with the control group,the expression of cleaved caspase-8 protein in the H₂O-₂ group increased significantly（P < 0.05）;compared with the H₂O-₂ group,the expression in the H₂O-₂+ acupunture serum group decreased（P < 0.05）;after adding SP600125,the expression decreased（P< 0.05）.Conclusion1.Shallow needling and deep needling "Huantiao"（GB30）can improve the function of nerve movement and conduction,accelerate the recovery of nerve structure,and the effect of deep needling is better than that of shallow needling.It shows that acupuncture can improve the function of nerve movement and conduction,and repair the structure of tissue;acupuncture can improve the ability of antioxidation and reduce oxidative damage,and the effect of deep acupuncture is more significant.2.Acupuncture can regulate JNK signal pathway,inhibit c-Jun phosphorylation,increase the ratio of Bcl-2 / Bax gene and protein,decrease the expression of Caspase-8 and caspase-3 gene and activated protein,inhibit the apoptosis of nerve cells,and have better effect of deep acupuncture.It is suggested that acupuncture can inhibit neuronal apoptosis by regulating JNK signaling pathway.3.The expression of S100 in the local tissue of sciatic nerve injury was decreased,and the expression of S100 in the local tissue of injury was increased by acupuncture.It is suggested that acupuncture can promote the proliferation and maturation of Schwann cells.4.Acupuncture serum can promote the proliferation of RSC96 cells with oxidative damage;acupuncture serum and sp600125 can inhibit apoptosis by regulating JNK signal pathway;the mechanism of acupuncture serum inhibiting apoptosis of RSC96 cells with oxidative damage may be realized by regulating JNK signal pathway.