| Objective To observe the effects of Jiaji electroacupuncture combined with treadmill training on tibial bone mineral density,volume and number of proximal tibial vessels,VEGF expression in tibial bone marrow,spinal cord and sciatic nerve,as well as VEGFm RNA and protein expression in tibial bone tissue in rats with sciatic nerve transection injury,to explore the mechanism of Jiaji electroacupuncture combined with treadmill training on tibia reconstruction in rats with sciatic nerve amputation injury.Methods 180 SD rats were randomly divided into 5 groups: control group 2(normal control group and model control group),treatment group 3(Jiaji electroacupuncture group,treadmill training group,Jiaji electroacupuncture combined with treadmill training group,hereinafter referred to as the combination group),36 rats in each group.They were divided into 3 subgroups according to treatment time points: 2 weeks,4 weeks and 8 weeks,with 12 animals in each subgroup.Normal control group: no intervention;model control group: sciatic nerve severing injury and suture;Jiaji electro-acupuncture group: taking " Waist Jiaji Point" for electro-acupuncture treatment;treadmill group: starting on the third day after successful modeling Treadmill training;combined group: treadmill training and Jiaji electro-acupuncture.After the corresponding intervention time points,the rats in each group were scanned by Micro-CT to analyze the change trend of tibial bone mineral density;the proximal tibial angiography was used for quantitative analysis of tibial blood vessels; RT-PCR and Western Blot were used to determine the tibial bone tissue.VEGFm RNA and protein expression;ELISA to determine the change of VEGF concentration in tibial bone marrow;immunohistochemical method to determine the expression of VEGF in spinal cord and sciatic nerve respectively.Result1.Micro-CT determination of tibial bone mineral density After 2,4,and 8 weeks of intervention,the BMD of the tibia of the rats in the three treatment groups was higher than that of the model control group at the same time point,and lower than that of the normal control group(P<0.05);at each time point,the BMD of the tibia of the rats in the combined group was higher than that of the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);at each time point,the BMD of the tibia of the Jiaji electro-acupuncture group was higher than that of the treadmill training group(P<0.05);the BMD of the tibia of the rats in each group was the highest at the8-week time point.2.Microfil perfusion proximal tibia angiography After 2,4,and 8 weeks of intervention,compared with the normal control group,severe blood vessel damage was observed in the proximal tibia of the model control group,and the volume and number of blood vessels decreased over time;after intervention,significantly increased microscopic blood vessels were observed in the proximal tibia of the three treatment groups;after 2 weeks of intervention,the volume and number of proximal tibia blood vessels in the combination group and the electroacupuncture group were higher than those in the normal control group and the treadmill group(P<0.05);and the volume and number of blood vessels in the proximal tibia of the normal control rats were higher than those in the treadmill training group(P<0.05);after 4 and 8 weeks of intervention,significantly increased microscopic blood vessels were observed in the proximal tibia of the three treatment groups,and the volume and number of blood vessels were higher than those in the normal control group and the model control group(P<0.05);at each time point,the volume and number of proximal tibia blood vessels in the combined group were higher than those in the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);and the volume and number of proximal tibia blood vessels in the Jiaji electro-acupuncture group were higher than those in the treadmill training group(P<0.05);the volume and number of blood vessels in the proximal tibia of rats in each treatment group were the highest at 8 weeks.3.VEGF m RNA and protein expression in tibial bone tissue(1)RT-PCR fluorescence quantitative analysis:After 2,4 and 8 weeks of intervention,compared with the normal control group,the expression of VEGF m RNA in the tibia bone tissue of the model control group showed a decreasing trend over time;at each time point,the expression of VEGF m RNA in the tibia bone tissue of the three treatment groups was higher than that of the model control group(P<0.05);and the expression of VEGF m RNA in the tibia bone tissue of the rats in the combination group was better than that in the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);at each time point,the relative expression of VEGF m RNA in the tibia bone tissue of the treadmill training group was higher than that of the Jiaji electro-acupuncture group(P<0.05);the relative expression of VEGF m RNA in rat tibia bone tissue in each group reached the peak at 2weeks,and showed a downward trend at 4 and 8 weeks.(2)Western blot gray value analysis:After 2,4 and 8 weeks of intervention,compared with the normal control group,the expression of VEGF m RNA in the tibia bone tissue of the model control group showed a decreasing trend over time;at each time point,the expression of VEGF protein in the tibia bone tissue of the three treatment groups was higher than that of the model control group(P<0.05);and the expression of VEGF protein in the tibia bone tissue of the rats in the combination group was better than that in the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);at each time point,the relative expression of VEGF protein in the tibia bone tissue of the treadmill training group was higher than that of the Jiaji electro-acupuncture group(P<0.05);the relative expression of VEGF protein in rat tibia bone tissue in each group reached the peak at 2 weeks,and showed a downward trend at 4 and 8 weeks.4.Changes of VEGF Concentration in Tibial Bone Marrow by ELISA After 2,4 and 8 weeks of intervention,the concentration of VEGF in the tibial bone marrow of the three treatment groups was higher than that of the normal control group and the model control group(P<0.05);and the concentration of VEGF in the tibial bone marrow of the rats in the normal control group was higher than that in the model group(P<0.05);at each time point,the concentration of VEGF in the tibia bone marrow of the combined group was better than that of the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);and the concentration of VEGF in the tibia bone marrow of the treadmill training group was higher than that of the Jiaji electro-acupuncture group(P<0.05);the concentration of VEGF in the tibia bone marrow of rats in each group reached the peak at 2weeks,and showed a downward trend at 4 and 8 weeks.5.VEGF expression in spinal cord and sciatic nerve(1)Spinal cord segment: After 2,4 and 8 weeks of intervention,compared with the normal control group,the expression of VEGF in the spinal cord of the rats in the model control group decreased over time;at each time point,the expression of VEGF in the spinalcord of the rats in the three treatment groups was higher than that in the normal control group and the model control group(P<0.05);and the expression of VEGF in the spinal cord of the rats in the combination group was better than that in the simple Jiaji electro-acupuncture group and the treadmill training group at the same time point(P<0.05);at each time point,the expression of VEGF in the spinal cord of the rats in the Jiaji electro-acupuncture group was higher than that in the treadmill training group(P<0.05);the expression of VEGF in the spinal cord of rats in each group reached a peak at 2 weeks,and showed a downward trend at 4 and 8 weeks.(2)Sciatic nerve: After 2,4 and 8 weeks of intervention,compared with the normal control group,the expression of VEGF in the sciatic nerve of the rats in the model control group decreased over time;at each time point,the expression of VEGF in the sciatic nerve of the rats in the three treatment groups was higher than that in the normal control group and the model control group(P<0.05);and the expression of VEGF in the sciatic nerve of the rats in the combination group was better than that in the simple Jiaji electro-acupuncture group and the treadmill training group(P<0.05);at each time point,the expression of VEGF in the sciatic nerve of the rats in the Jiaji electro-acupuncture group was higher than that in the treadmill training group(P<0.05);the expression of VEGF in the sciatic nerve of rats in each group reached a peak at 2 weeks,and showed a downward trend at 4 and 8 weeks.Conclusion1.Jiaji electroacupuncture,treadmill training,Jiaji electroacupuncture combined with treadmill training can improve tibial bone mineral density,increase the volume and number of proximal tibia blood vessels,and up-regulate VEGF expression in tibial bone marrow,spinal cord and sciatic nerve in rats with sciatic nerve transection injury.The expression of m RNA and protein in tibial bone tissue was better in the combined group than in the simple Jiaji electro-acupuncture group and the treadmill training group.2.Jiaji electroacupuncture combined with treadmill training improves tibial bone mineral density and increases the volume and number of proximal tibial blood vessels,which may be related to the increase of VEGF m RNA and protein expression,which may be one of the mechanisms of tibial reconstruction after peripheral nerve injury. |
