| Background: Chronic low back pain is a spinal joint disease with a high incidence in modern society.It is often accompanied by pain and dysfunction.Its pathogenesis is closely related to lumbar degeneration.Spinal manipulative therapy has a definite clinical effect in treating this disease and is widely applicable,but the underlying mechanism of tuina intervention is still unclear.Previous studies have indicated that the inflammatory microenvironment plays an important role in the occurrence and development of chronic pain and affects upstream signaling pathways.Among them,the PKCε-TRPV1 signaling pathway has a coexistence relationship with some inflammatory factors in the process of pain.In this study,an external linked fixation system was used to restrict the movement of rats’ lumbar spine,simulated lumbar degeneration,and induced a rat model of chronic low back pain(CLBP).Then we used the spine stiffness test,pain behavior test,Hematoxylin-eosin staining(HE),Cat Walk gait analysis system,Enzyme-Linked Immuno Sorbent Assay(ELISA),Immunofluorescence(IF),Western Blot(WB)and other research methods to observe the analgesic effect of spinal manipulative therapy on CLBP model rats,changes in gait parameters,changes in the inflammatory microenvironment of local tissues of the waist,and the effects of PKCε and TRPV1 receptors and its phosphorylation expression in DRG neurons,which for exploring the related analgesic mechanisms of spinal manipulative therapy for the treatment of chronic low back pain.Part I: The analgesic effect of spinal manipulation on CLBP model rats Objective: To explore the effects of external linked fixation system on the stiffness of the rats’ lumbar spine;to explore the changes in pain behavior of rats after external linked fixated;to explore the analgesic effect of spinal manipulation on CLBP model rats and the changes in the morphology of rat intervertebral discs.Methods: This part is divided into two sections.Section 1: Observe the effect of external linked fixation steel plate on the stiffness of the lumbar spine of rats.16 SD rats were randomly divided into 2 groups,8 in each group,which were model group and model control group.The changes in the spine stiffness of two groups of rats were observed at the end of the first week and the second week after operation.Section 2:(1)24 SD rats were randomly divided into 3 groups,8 in each group,which were the blank group,sham operation group and CLBP model group.After the model was completed,they were feeded and observed for 2 weeks.After 2 weeks,measured the Paw withdrawl threshold(PWT)and the Paw withdrawl latency(PWL)of the 1st,3rd,7th,10 th,and 14 th days after formation of the model.(2)32 SD rats were randomly divided into 4 groups,8 in each group,which were the blank group,sham operation group,CLBP model group and tuina treatment group.After the model was completed,they were feeded and observed for 2 weeks.After 2 weeks,spinal manipulative intervention was applied to the tuina treatment group.PWT and PWL were measured at 1st,3rd,7th,10 th,and 14 th days after the intervention.After the end of the experimental period,the rats’ intervertebral discs were taken out and used HE staining to observe the morphological changes of the intervertebral discs.Results:Section 1: After the model group and the model control group were fixed for 7days,the spine stiffness of two groups of rats were significantly different,and the difference was statistically significant(P < 0.05);after 14 days,the difference in spine stiffness tended to increase,and the difference was statistically significant(P < 0.01).There was no statistically significant difference in the stiffness of the spine of the model control group 14 days later than immediately after the model was completed(P > 0.05).Section 2:(1)There was no statistically significant in PWT and PWL between the three groups of rats before modeling(P > 0.05).The PWT and PWL of the CLBP model group decreased significantly after modeling,and there were statistically significant differences between the blank group and the sham operation group at each time point(P < 0.01).(2)The PWT and PWL of rats in the tuina treatment group showed an overall upward trend after intervention.At 10 th day to 14 th day of intervention,the difference was statistically significant between the tuina treatment group and the CLBP model group(P < 0.01).(3)Observation of rat intervertebral disc cell morphology under light microscope revealed that the CLBP model group and the tuina treatment group had fibrocartilaginous degeneration and appeared a large number of inflammatory cells.The blank group and the sham operation group had regular intervertebral disc cell morphology and no significant difference.Conclusion: Long-term external linked fixation of the lumbar spine in rats can generate spinal degeneration,reduce the threshold of mechanical stimulation and the latency of thermal stimulation.Spinal manipulation can improve the mechanical and thermal hyperalgesia induced by this model.It has not been found that spinal manipulation can affect the morphology of intervertebral discs in the CLBP model rats.Part Ⅱ: Analysis of Cat Walk gait parameters of spinal manipulation on CLBP model ratsObjective: To explore the effects of spinal manipulation on the gait parameters of CLBP model rats.Methods: 32 SD rats were randomly divided into 4 groups,8 in each group,which were the blank group,sham operation group,CLBP model group and tuina treatment group.After the model was completed,they were feeded and observed for 2 weeks.After 2 weeks,spinal manipulative intervention was applied to the tuina treatment group.On the 1st,3rd,7th,10 th and 14 th days after the intervention,the Cat Walk gait analysis system was used to detect the standing phase duration(stands),the swinging phase duration(swings),the swing speed and walking duration(duration)of four groups of rats.Results: There were no statistically significant differences in the standing phase duration,walking phase duration,swing speed,walking duration of four groups of rats before modeling(P > 0.05).The differences of standing phase duration of the both hind limbs in the CLBP model group were statistically significant compared with the blank group and the sham operation group after formation of the model(P < 0.01).By the 14 th day after the intervention,the differences of standing phase duration of the both hind limbs in the tuina treatment group were statistically significant compared with the CLBP model group(P < 0.05);The differences of standing phase duration of the both hind limbs in the CLBP model group were statistically significant compared with the blank group and the sham operation group after formation of the model(P < 0.01).By the 7th day after the intervention,the difference of swinging phase duration of the left hind limb in the tuina treatment group was statistically significant compared with the CLBP model group(P < 0.05),by the 10 th and 14 th days after the intervention,the differences of swinging phase duration of the both hind limbs in the tuina treatment group were statistically significant compared with the CLBP model group(P < 0.05);The differences of swing speed of the both hind limbs in the CLBP model group were statistically significant compared with the blank group and the sham operation group after formation of the model(P < 0.01).By the 10 th day after the intervention,the differences of the both hind limbs in the tuina treatment group were statistically significant compared with the CLBP model group(P < 0.05),by the 14 th day after the intervention,the differences of the both hind limbs in the tuina treatment group were statistically significant compared with the CLBP model group(P < 0.01);The differences of walking duration of rats in the CLBP model group were statistically significant compared with the blank group and the sham operation group after formation of the model(P < 0.01).By the 10 th and 14 th days after the intervention,the differences of walking duration of rats in the tuina treatment group were statistically significant compared with the CLBP model group(P < 0.01).Conclusion: The CLBP model rats have different degrees of changes in the standing phase duration,swinging phase duration,swing speed and walking duration compared with the blank group and the sham operation group,which suggest that the CLBP model rats have different degrees of dysfunction after formation of the model.Spinal manipulation intervention can improve this phenomenon.Part Ⅲ The effect of spinal manipulation on the expression of PKCε-TRPV1 and related pro-inflammatory factors on CLBP model ratsObjective: To observe the effect of spinal manipulation on the local inflammatory microenvironment on CLBP model rats;to explore the role of TRPV1 in mediating rats’ hyperpathia;to explore the analgesic mechanism of spinal manipulation by regulating PKCε-TRPV1 signaling pathway;to explore the blocking effect of pain with spinal manipulation.Methods: This part is divided into three sections.Section 1: 32 SD rats were randomly divided into 4 groups,8 in each group,which were the blank group,sham operation group,CLBP model group and tuina treatment group.After the model was completed,they were feeded and observed for 2weeks.After 2 weeks,spinal manipulative intervention was applied to the tuina treatment group.The animals were executed after 2 weeks of intervention,the local muscle tissue of the waist was taken out and the content of calcitonin gene-related peptide(CGRP),nerve growth factor(NGF),substance P(SP)and serotonin(5-HT)in the tissue were detected by ELISA.Section 2: 18 SD rats were randomly divided into 3 groups,6 in each group,which were the blank group,the plantar injection of normal saline group,and the plantar injection of Capsaicin group.The blank group was not treated,and the other two groups were injected with 0.9% saline and Capsaicin on the plantar.The PWT and PWL of three groups of rats were measured on the 1st,3rd,5th,and 7th days after the injection.The animals were executed after 7 days of injection,the rats’ L4-L6 dorsal root ganglions were taken out and the protein contents of TRPV1 in the L4-L6 dorsal root ganglions of rats were detected by Western blot.Section 3:(1)32 SD rats were randomly divided into 4 groups,8 in each group,which were the blank group,sham operation group,CLBP model group and tuina treatment group.After the model was completed,they were feeded and observed for 2weeks.After 2 weeks,spinal manipulative intervention was applied to the tuina treatment group.The animals were executed after 2 weeks of intervention,the rats’ L4-L6 dorsal root ganglions were taken out,and the expression of PKCε and TRPV1 and the contents of PKCε,TRPV1 and phospho-TRPV1 protein in the L4-L6 dorsal root ganglions of rats were detected by Western blot and tissue immunofluorescence technique.(2)12 SD rats were randomly divided into 2 groups,6 in each group,which were the CLBP model group,model plus injection of Sotrastaurin group.After the model was completed,they were feeded and observed for 2 weeks.Sotrastaurin was intraperitoneally injected into one group of rats.The PWT and PWL of two groups of rats were measured on the 1st,3rd,7th,and 10 th days after the injection.The animals were executed after 10 days of injection,the rats’ L4-L6 dorsal root ganglions were taken out and the contents of TRPV1 and phospho-TRPV1 protein in the L4-L6 dorsal root ganglions of rats were detected by Western blot.Results:Section 1:(1)The protein contents of CGRP,NGF,SP,5-HT in the DRG neurons of the CLBP model group and the tuina treatment group was significantly higher than that of the blank group and the sham operation group,the differences of the first two groups were statistically significant compared with the latter two groups(P < 0.01).(2)The protein contents of CGRP,NGF and SP in DRG neurons in the tuina treatment group was significantly lower than that in the CLBP model group,and the differences between the groups were statistically significant(P <0.01);The protein contents of 5-HT in DRG neurons in the tuina treatment group was significantly lower than the CLBP model group,and the difference between the groups was statistically significant(P <0.05).Section 2:(1)There was no significant difference in PWT and PWL among the three groups of rats before the injection(P > 0.05).The PWT and PWL of the rats in the plantar injection of Capsaicin group decreased significantly from the first day of injection until the end of the period.On the 1st,3rd,5th and 7th days after injection,the differences were statistically significant compared with the blank group and the plantar injection of NS group(P < 0.01).The pain threshold of rats in the plantar injection of NS group also indicated a downward trend.On the 1st and 7th days after the injection,the difference of PWT was statistically significant compared with the blank group(P < 0.05).On the 3rd day after the injection,the difference of PWT was statistically significant compared with the blank group(P < 0.05).(2)The protein contents of TRPV1 in DRG neurons of rats in the plantar injection of Capsaicin group were significantly higher than those of the blank group and the plantar injection of NS group,and the difference was statistically significant(P <0.01).The protein contents of TRPV1 in the DRG neurons of rats in the plantar injection of NS group was slightly higher than that of the blank group,the difference was no statistically significant(P >0.05).Section 3:(1)The PKCε and TRPV1 receptors in the DRG neurons of the blank group and the sham operation group were slightly distributed and expressed,but not active.Compared with the previous two groups,the expressions in CLBP model group and the tuina treatment group were more obvious.Compared with the CLBP model group,the expressions of PKCε and TRPV1 receptors in the tuina treatment group had a significant inhibitory trend.(2)Compared with the blank group,the protein contents of PKCε,TRPV1 and p-TRPV1 in DRG neurons in the sham operation group were no statistically significant(P > 0.05);The protein contents of PKCε,TRPV1 and p-TRPV1 in DRG neurons in the CLBP model group and the tuina treatment group were significantly higher than those in the blank group and the sham operation group,the differences of the first two groups were statistically significant compared with the latter two groups(P < 0.01);The protein contents of PKCε and p-TRPV1 in DRG neurons in the tuina treatment group was lower than those in the CLBP model group,and the differences between the groups were statistically significant(P < 0.01);the protein content of TRPV1 in DRG neurons in the tuina treatment group was lower than that in the CLBP model group,and the difference between the groups was statistically significant(P < 0.05).(3)There were no significant differences in PWT and PWL between the two groups of rats before the injection(P > 0.05).The PWT and PWL in the intraperitoneal injection of Sotrastaurin group rats were increased significantly from 7th day after injection until the end of the period.On the 7th day after the injection,compared with the CLBP model group,the difference of PWT was statistically significant(P < 0.01),and compared with the CLBP model group,the difference of PWL was statistically significant(P < 0.05);On the 10 th day after the injection,compared with the CLBP model group,the differences of PWT and PWL were statistically significant(P<0.01).(4)The protein contents of TRPV1 and p-TRPV1 in DRG neurons of rats in the intraperitoneal injection of Sotrastaurin group was significantly lower than those in the CLBP model group,and the differences was statistically significant(P < 0.01).Conclusion: The expression of local inflammatory factors increased in CLBP model rats,spinal manipulation can improve the local inflammatory microenvironment of the waist;TRPV1 mediates mechanical pain and thermal hyperalgesia in rats,and spinal manipulation intervention can reduce protein contents of PKCε,TRPV1 and phosphorylated TRPV1 in DRG neurons of CLBP model rats.Blocking PKCε had analgesic effects in CLBP model rats,and reduced the protein contents of TRPV1 and phosphorylated TRPV1 in DRG neurons.Spinal manipulation can produce analgesic effects similar to antagonist. |
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