| Objective1.To provide a quantitative basis for the clinical application and research of Lin’s bone massage manipulation by collecting the relevant mechanical parameters of the Lin’s pillow extension pressing manipulation for the treatment of lumbar disc herniation(LDH).2.To observe the differences in clinical efficacy of different compression frequencies of the cushion-pillow back extension compression method on LDH,and to compare it with traditional massage techniques,to objectively and scientifically evaluate the safety and effectiveness of the cushion-pillow back extension compression method in the treatment of LDH,and to explore the optimal treatment frequency of the cushion-pillow back extension compression method.3.Based on the Wnt/β-catenin signaling pathway,to explore the mechanism of pressing manipulation on the cartilage endplates of degenerated intervertebral discs in rats,so as to provide a potential treatment strategy for the lumbar disc degeneration-related diseases.Methods1.Ten participants with LDH who met the inclusion criteria were recruited in the Massage Department of Guangdong Provincial Hospital of Traditional Chinese Medicine.MFF multi-point membrane pressure testing system was selected to collect the dynamic graphics and the corresponding manipulative mechanical parameters of the Lin’s pillow extension pressing manipulation,including preload force,valley value,maximum pressing force,pressing time,duration.Before and after treatment,visual analogue scale(VAS)score was used to evaluate the analgesic effect of Lin’s pillow extension pressing manipulation in the treatment of LDH patients.2.One hundred and forty patients with LDH were randomly divided into four groups:traditional massage group(n=35)and Lin’s pillow extension pressing manipulation group(n=115).According to the different frequency of Lin’s pillow extension pressing manipulation,it was divided into three groups further,which were low frequency group(n=35),medium frequency group(n=35)and high frequency group(n=35).VAS,Oswestry disability index(ODI),lumbar range of motion(flexion,extension,left and right lateral flexion)were measured before and after the treatment(4 weeks),and 1-month follow-up.Also,adverse events occurred during treatment were recorded for the evaluation of safety.3.The rat model of lumbar intervertebral disc degeneration was established by forelimb amputation in rats and standing upright.The normal group was used as the control group.The rats were treated with pressing manipulation,sham manipulation after 5-months modeling for 60 days.After the intervention,HE staining and Safranin O-Fast Green staining were used to observe the pathological changes of cartilage endplate of degenerative intervertebral disc in each group,to evaluated the therapeutic effect of pressing manipulation on lumbar intervertebral disc degeneration rats.In addition,in order to explore the mechanism of pressing manipulation,the protein and mRNA expression levels of key molecules Wnt-3a,β-catenin,and GSK-3 β of Wnt/β-catenin signaling pathway and its downstream factor MMP-13 in cartilage endplate of degenerative intervertebral disc were detected by immunohistochemistry,Western Blot and qRT-PCR.Results1.The first section:The dynamic parameters of Lin’s pillow extension pressing manipulation for the treatment of LDH.The dynamic parameters of Lin’s pillow extension pressing manipulation in the treatment of LDH showed that the average preloading force was 402.00±17.58N,the operation time was 1.25s to 1.31s,the average valley value was 164.41±9.57N,the peak range of pressing was 592.64 ± 13.43N,the impact time was 0.15s to 0.18s,the impact velocity was 3736.70 ±190.72(N/s)and the impulses of 60.21 ±3.92(N·s).There was no significant difference in dynamic parameters among different participants(P>0.05).The VAS score of the participants before and after treatment was 5.93±0.53 and 3.19±0.68,respectively,and the difference of VAS score was significant(P<0.01).It showed that the Lin’s pillow extension pressing manipulation had a good analgesic effect on patients with LDH.When the skilled operator of Lin’s using the Lin’s pillow extension pressing manipulation,the dynamic parameters preloading force,the valley value,the peak of pressing,the impact velocity and the impulses were maintain in stable output,which was considered as the crucial reason to the analgesic effect.2.The second section:The clinical efficacy and safety of the Lin’s pillow extension pressing manipulation in the treatment of LDH.A total of 140 patients with LDH were included in the study,while 14 dropped out during the research,and finally a total of 126 patients completed the treatment and follow-up.(1)VAS score:The overall trend of VAS scores decreased after treatments in all groups,with the largest decrease in VAS scores after the first treatment,indicating that each group had immediate analgesic effects.At different observation points,there were significant differences in the low,medium and high frequency groups compared with the traditional massage group(P<0.05),respectively,indicating that different frequency of Lin’s pillow extension pressing manipulation was superior than the traditional massage group in relieving pain caused by LDH and the analgesic effect of the medium frequency group was better than that of the low and high frequency groups,with statistically significant differences(P<0.05).And the analgesic effect of the medium frequency group was better than that of the low and high frequency groups,with statistically significant differences(P>0.05).Also,the effect of immediate analgesia and analgesic maintaining in the medium frequency of pressing were the best.(2)ODI score:With the change of treatment time,the ODI scores of patients in each group showed different degrees of relief,and the differences were statistically significant(P<0.05)in the low,medium and high frequency groups compared with the traditional massage group,respectively.The differences were statistically significant(P<0.05)in the medium frequency group compared with the low and high frequency groups,and there was no statistical significance(P>0.05)between the low and high frequency group.This indicated that the curative effect of different frequency of Lin’s pillow extension pressing manipulation were more effective than the traditional massage group in improving the dysfunction of patients with LDH,among which the medium frequency group was significantly more effective.(3)Lumbar range of motion:The overall trend of lumbar anterior flexion,posterior extension,and left and right lateral flexion mobility of patients in each group increased after treatment,indicating that the function of multi-directional lumbar mobility was improved in each group with time change.While the medium frequency group showed significant difference when compared to the other three groups(P<0.05).The effect of low frequency group and high frequency group was similar to that of traditional massage group(P>0.05),and there was no significant difference between low frequency group and high frequency group(P>0.05).The results indicated that the medium frequency group had a significant effect in improving the movement function of the patients in all directions of the lumbar.(4)Clinical efficacy:There was no significant difference in the overall clinical efficacy of each group at the end of the treatment course(P>0.05).The clinical curative effects of each group were compared in pairs,the middle frequency group was compared with the traditional massage group,the differences were statistically significant(P<0.05),and the clinical curative effects were comparable among the other groups(P>0.05).3.The third part:The mechanism of pressing manipulation for the endplate of IVDD rats by Wnt/β-catenin signaling pathway.(1)H&E staining:Compared with the control group,the number of notochord cells in the nucleus pulposus appeared significantly reduced in the model group,the lamellar structure of fibrous annulus was unclear,disordered and obvious fracture.The structure and boundary of cartilage endplate was unclear,and chondrocytes were hypertrophic and widely distributed.In pressing group,after intervention,compared with the model group,the overall structure of intervertebral disc tissue was more complete,the nucleus pulposus notochord cells were abundant,and the structure of annulus fibrosus lamina was clear with a small amount of local fissures.The cartilage endplate structure was clear and complete and hypertrophic chondrocytes were significantly reduced.While in the sham pressing group,no significant improvement was discovered compared with the model group.(2)Safranin O-Fast Green staining:Compared with the control group,the defect of cartilage endplate tissue was detected in the model group,the Saffron O staining became lighter,while the cartilage endplate cells showed hypertrophy and vacuole,and the range of Fast Green staining was significantly increased and deepened in color,indicating that the collagen contenting in cartilage endplate decreased and degenerated obviously.After pressing intervention,compared with the model group,the structure and morphology of cartilage endplate was more complete,the Saffron O staining was deeper and uniform,and the number of hypertrophic chondrocytes decreased significantly.There was no significant improvement in the sham pressing group compared with the model group.(3)Immunohistochemistiy:Compared with the control group,the positive expression of Wnt3a,β-catenin and MMP-13 in the cartilage endplate of the model group was significantly increased,showing a large number of brown-stained positive cells,and the expression of GSK-3β decreased and was negative in the cartilage endplate.However,the protein expression of Wnt3a,β-catenin and MMP-13 decreased significantly after treated with pressing,with GSK-3β protein increased.The expression level of the above proteins in the sham pressing group was not significantly different from that in the model group.(4)Western Blot:Compared with the control group,the expression of Wnt3a,β-catenin and MMP-13 protein in model group were significantly increased(P<0.01),and the expression of GSK-3β decreased(P<0.01).Compared with the model group,the protein expression of Wnt3a,β-catenin and MMP-13 decreased significantly(P<0.01),and the expression of GSK-3β increased(P<0.01)in the pressing group.These protein expression in the sham pressing group were not significantly different from those in the model group(P>0.05).These changes were consistent with the results of immunohistochemistry.(5)qRT-PCR:Compared with the control group,the expression of Wnt3a,β-catenin and MMP-13mRNA in the cartilage endplate of the model group was significantly up-regulated(P<0.01),while the expression of GSK-3β mRNA was significantly decreased(P<0.01).Compared with the model group,the expression of Wnt3a,β-catenin and MMP-13 mRNA decreased significantly(P<0.01),while the expression of GSK-3βincreased significantly(P<0.05)after pressing intervention in the pressing group.The expression level of mRNAs in the sham pressing group had no difference from those in the model group(P>0.05).The expression of the above genes in the cartilage endplate of each group were consistent with the corresponding protein level.Conclusion1.When the skilled operator of Lin’s using the Lin’s pillow extension pressing manipulation,the dynamic parameters preloading force,the valley value,the peak of pressing,the impact velocity and the impulses were maintain in stable output,which was considered as the crucial reason to the analgesic effect.2.Different frequency of Lin’s pillow extension pressing manipulation had greater advantages than traditional massage manipulation in relieving pain and dysfunction,as well as improving the movement function of lumbar of patients with LDH,especially in the medium frequency group(11-15 times),and the safety was satisfied.3.Pressing manipulation could delay the degree of cartilage endplate degeneration of lumbar intervertebral disc by effecting the Wnt/β-catenin signaling pathway,and then protect the degenerative lumbar intervertebral disc. |
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