[1]张力,张华,李平,等.中医整脊棘突旁指压法干预大鼠膝骨关节炎的效果及作用机制研究[J].中医正骨,2025,37(03):14-22,38.
　ZHANG Li,ZHANG Hua,LI Ping,et al.Efficacy and mechanism of traditional Chinese medicine chiropractic paraspinous thumb-pressing therapy against knee osteoarthritis in rats:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(03):14-22,38.
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中医整脊棘突旁指压法干预大鼠膝骨关节炎的效果及作用机制研究()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年03期
页码:
14-22,38
栏目:
基础研究
出版日期:
2025-03-20

文章信息/Info

Title:
Efficacy and mechanism of traditional Chinese medicine chiropractic paraspinous thumb-pressing therapy against knee osteoarthritis in rats:an experimental study
作者:
张力; 张华; 李平; 高嵩; 柯广娟; 屈留新
东南大学附属中大医院,江苏 南京 210009
Author(s):
ZHANG Li; ZHANG Hua; LI Ping; GAO Song; KE Guangjuan; QU Liuxin
Zhongda Hospital Affiliated to Southeast University,Nanjing 210009,Jiangsu,China
关键词:
骨关节炎; 膝;  中医整脊;  指压法;  疼痛;  敏化;  瞬时受体电位香草素受体1;  神经节; 脊;  神经元;  滑膜;  成纤维细胞;  串话;  大鼠
Keywords:
osteoarthritis; knee;  traditional Chinese medicine chiropractic;  thumb pressing manipulation;  pain;  sensitization;  transient receptor potential vanilloid 1;  ganglia; spinal;  neurons;  synovial membrane;  fibroblasts;  crosstalk;  rats
摘要:
目的:探讨中医整脊棘突旁指压法干预大鼠膝骨关节炎(knee osteoarthritis,KOA)的效果及作用机制。方法:将40只SD大鼠随机分为空白组、模型组、低强度组、中强度组和高强度组,每组8只。模型组、低强度组、中强度组和高强度组大鼠均采用碘乙酸钠关节腔注射建立双侧KOA模型,空白组大鼠双侧膝关节腔注射等量生理盐水。造模14 d后,采用小动物计力指压器于L3～L5棘突旁,对低、中、高强度组大鼠分别施以4 N、8 N、16 N的垂直向下压力,模拟不同力度的棘突旁指压法; 每次按压2 s,每处连续按压2 min; 每隔1 d施术1次,干预28 d; 空白组和模型组不进行任何干预。分别于干预1 d、7 d、14 d、21 d、28 d,采用von Frey电子测痛仪测定大鼠机械刺激缩足阈值(paw withdrawal mechanical threshold,PWMT)。干预28 d后,采用ELISA试剂盒检测大鼠血清中神经生长因子(nerve growth factor,NGF)、降钙素基因相关肽(calcitonin gene related peptide,CGRP)含量; 采用钙成像技术检测大鼠L3背根神经节神经元Ca2+通量; 采用Western blot法检测大鼠L3背根神经节组织中瞬时受体电位香草素受体1(transient receptor potential vanilloid 1,TRPV1)、脂蛋白磷酸酶2B(protein phosphatases 2B,PP2B)、钙调蛋白(calmodulin,CaM)的蛋白表达水平; 分别采用HE染色试剂盒、Masson染色试剂盒、天狼星红染色试剂盒对大鼠膝关节滑膜组织进行染色,观察滑膜组织的炎性细胞浸润和纤维化情况; 采用Western blot法检测大鼠膝关节滑膜组织中白细胞介素(interleukin,IL)-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、转化生长因子(transforming growth factor,TGF)-β、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的蛋白表达水平。结果:①PWMT测定结果。空白组、模型组、低强度组大鼠PWMT随时间变化均基本保持不变(F=0.885,P=0.487; F=0.339,P=0.849; F=0.555,P=0.697),中、高强度组大鼠PWMT随时间变化均呈上升趋势(F=8.222,P=0.000; F=9.844,P=0.000)。干预21 d、28 d,中、高强度组大鼠PWMT均高于模型组(干预21 d:P=0.002,P=0.001; 干预 28 d:P=0.003,P=0.000)和低强度组(干预21 d:P=0.022,P=0.011; 干预28 d:P=0.044,P=0.003),高强度组大鼠PWMT与中强度组比较,差异均无统计学意义(P=0.774,P=0.273)。②血清疼痛介质含量测定结果。中、高强度组大鼠血清NGF、CGRP含量均低于模型组(NGF含量:P=0.003,P=0.000; CGRP含量:P=0.000,P=0.000); 中强度组大鼠血清NGF含量与低强度组比较,差异无统计学意义(P=1.000),血清CGRP含量低于低强度组(P=0.000); 高强度组大鼠血清NGF、CGRP含量均低于低强度组(P=0.027,P=0.000); 高强度组大鼠血清NGF、CGRP含量与中强度组比较,组间差异均无统计学意义(P=1.000,P=1.000)。③L3背根神经节神经元Ca2+通量检测结果。低、中、高强度组大鼠L3背根神经节神经元Ca2+平均荧光强度均高于模型组(P=0.001,P=0.000,P=0.000),中、高强度组大鼠L3背根神经节神经元Ca2+平均荧光强度均高于低强度组(P=0.003,P=0.016),高强度组大鼠L3背根神经节神经元Ca2+平均荧光强度与中强度组比较,差异无统计学意义(P=0.361)。④L3背根神经节组织中TRPV1、PP2B、CaM的蛋白表达水平检测结果。中、高强度组大鼠L3背根神经节组织中TRPV1的蛋白相对表达量均高于模型组和低强度组(模型组:P=0.002,P=0.000; 低强度组:P=0.047,P=0.014),PP2B、CaM的蛋白相对表达量均低于模型组和低强度组(模型组:P=0.011,P=0.000; P=0.000,P=0.000; 低强度组:P=0.043,P=0.033; P=0.000,P=0.000); 高强度组大鼠L3背根神经节组织中TRPV1、PP2B、CaM的蛋白相对表达量与中强度组比较,组间差异均无统计学意义(P=1.000,P=1.000,P=1.000)。⑤膝关节滑膜组织病理学观察结果。HE染色显示,模型组大鼠滑膜组织中呈现明显的炎性细胞浸润,低、中、高强度组大鼠滑膜组织中炎性细胞浸润较模型组减轻,且中、高强度组减轻明显; Masson染色显示,模型组大鼠滑膜组织中胶原沉积较空白组明显增加,低、中、高强度组大鼠滑膜组织中胶原沉积较模型组减少,且中、高强度组减少明显; 天狼星红染色显示,模型组大鼠滑膜组织中Ⅱ型胶原明显减少,低、中、高强度组大鼠滑膜组织中Ⅱ型胶原较模型组增多,且中、高强度组增多明显。⑥膝关节滑膜组织中IL-1β、TNF-α、TGF-β、α-SMA的蛋白表达水平检测结果。中、高强度组大鼠滑膜组织中IL-1β、TNF-α、TGF-β、α-SMA的蛋白相对表达量均低于模型组和低强度组(模型组:P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000; 低强度组:P=0.004,P=0.002,P=0.003,P=0.000; P=0.005,P=0.008,P=0.007,P=0.004); 高强度组大鼠滑膜组织中IL-1β、TNF-α、TGF-β、α-SMA的蛋白相对表达量与中强度组比较,组间差异均无统计学意义(P=0.116,P=0.083,P=0.224,P=0.341)。结论:中医整脊棘突旁指压法干预大鼠KOA,能够减轻机械性疼痛敏化和滑膜炎症,且作用效果与力度有关; 其作用机制可能与其能引起背根神经节神经元TRPV1脱敏及影响背根神经节神经元与滑膜成纤维细胞间串话有关。
Abstract:
Objective:To observe the effects of traditional Chinese medicine(TCM)chiropractic paraspinous thumb-pressing therapy against knee osteoarthritis(KOA)in rats,and to explore its underlying mechanism.Methods:Forty SD rats were selected and randomized into blank group,model group,low-intensity group,medium-intensity group and high-intensity group,with 8 ones in each group.All rats but the ones in blank group were modeled by using bilateral knee intra-articular injection of sodium iodoacetate for inducing KOA,while the ones in blank group by using injection of the same dosage of normal saline at the corresponding sites.Fourteen days after successful modeling,the rats in the low-,medium-,and high-intensity groups were subjected to 4 N,8 N,and 16 N vertical downward pressures,respectively,beside the L3-L5 spinous processes by using a force-measuring finger pressure device for small animals to simulate the paraspinous thumb-pressing manipulation with different intensities,once every other day,2 seconds per time and 2 minutes per site for consecutive 28 days; while the ones in blank group and model group were not given any intervention.The paw withdrawal mechanical threshold(PWMT)of rats were tested using a Von Frey electronic algometer after 1-,7-,14-,21- and 28-day intervention,respectively.After 28-day intervention,the levels of nerve growth factor(NGF)and calcitonin gene related peptide(CGRP)in serum were detected by using ELISA kit,and the Ca2+ flux in L3 dorsal root ganglion(DRG)neurons was detected by using Calcium imaging.Furthermore,the protein expression levels of transient receptor potential vanilloid 1(TRPV1),protein phosphatases 2B(PP2B)and calmodulin(CaM)in the L3 DRG tissues were detected using Western blot.After that,the rat knee synovial tissues were harvested and stained with HE staining kit,Masson staining kit,and Sirius red staining kit,respectively,to observe the inflammatory cell infiltration and fibrosis,and the protein expression levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),transforming growth factor(TGF)-β,and α-smooth muscle actin(α-SMA)in the knee synovial tissues were detected using Western blot.Results:①The measuring results of PWMT.The PWMT remained essentially unchanged over time in blank group,model group,and low-intensity group(F=0.885,P=0.487; F=0.339,P=0.849; F=0.555,P=0.697),while that presented an upward trend over time in medium- and high-intensity groups(F=8.222,P=0.000; F=9.844,P=0.000).After 21- and 28-intervention,the PWMT was higher in medium- and high-intensity groups compared to model group and low-intensity group(21-intervention:P=0.002,P=0.001; P=0.022,P=0.011; 28-intervention:P=0.003,P=0.000; P=0.044,P=0.003),while,it was not significantly different from each other between high-intensity group and medium-intensity group(P=0.774,P=0.273).②The serum level of pain medi-ator.The serum levels of NGF and CGRP were lower in medium- and high-intensity groups compared to model group(NGF:P=0.003,P=0.000; CGRP:P=0.000,P=0.000).The serum level of CGRP was lower in medium-intensity group compared to low-intensity group(P=0.000),while,there was no statistical difference in the serum level of NGF between the 2 group(P=1.000).The serum levels of NGF and CGRP were lower in high-intensity group compared to low-intensity group(P=0.027,P=0.000),while,they were not significantly different from each other between high-intensity group and medium-intensity group(P=1.000,P=1.000).③The Ca2+ flux in L3 DRG neurons.The average fluorescence intensity of Ca2+ in L3 DRG neurons was higher in low-,medium-,and high-intensity groups compared to model group(P=0.001,P=0.000,P=0.000),and was higher in medium- and high-intensity groups compared to low-intensity group(P=0.003,P=0.016),while,it was not significantly different from each other between high-intensity group and medium-intensity group(P=0.361).④The protein expression levels of TRPV1,PP2B and CaM in the L3 DRG tissues.The relative protein expression level of TRPV1 in the L3 DRG tissues was higher,while that of PP2B and CaM was lower in medium- and high-intensity groups compared to model group and low-intensity group(model group:P=0.002,P=0.000; P=0.011,P=0.000; P=0.000,P=0.000; low-intensity group:P=0.047,P=0.014; P=0.043,P=0.033; P=0.000,P=0.000),while,there was no statistical difference in the relative protein expression levels of TRPV1,PP2B,and CaM between high-intensity group and medium-intensity group(P=1.000,P=1.000,P=1.000).⑤Pathological observation of knee synovial tissues.HE staining showed that,in model group,there was obvious inflammatory cell infiltration in the synovial tissues of rats,which was improved in low-,medium-,and high-intensity groups compared to model group,with a more pronounced improvement in medium- and high-intensity groups.Masson staining revealed that,compared to blank group,the collagen deposition in the synovial tissues significantly increased in rats of model group; compared to the model group,the collagen deposition decreased in low-,me-dium-,and high-intensity groups,with a more marked reduction in medium- and high-intensity groups.Sirius red staining showed that the typeⅡcollagen in the synovial tissues significantly decreased in rats of model group,while,compared to the model group,the typeⅡcollagen increased in low-,medium-,and high-intensity groups,with a more significant increase in medium- and high-intensity groups.⑥The protein expression levels of IL-1β,TNF-α,TGF-β and α-SMA in knee synovial tissues.The relative protein expression levels of IL-1β,TNF-α,TGF-β and α-SMA in knee synovial tissues were lower in medium- and high-intensity groups compared to model group and low-intensity group(model group:P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000; low-intensity group:P=0.004,P=0.002,P=0.003,P=0.000; P=0.005,P=0.008,P=0.007,P=0.004),while,they were not significantly different from each other between high-intensity group and medium-intensity group(P=0.116,P=0.083,P=0.224,P=0.341).Conclusion:The TCM chiropractic paraspinous thumb-pressing therapy can alleviate mechanical pain sensitization and synovial inflammation in KOA rats,and the effect exhibits intensity-dependence.It may work by inducing the desensitization of TRPV1 in DRG neurons and influencing the crosstalk between DRG neurons and synovial fibroblasts.

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备注/Memo

备注/Memo:
基金项目:东南大学附属中大医院中西医协同医学科研项目(2023zxyxt03)
通讯作者:屈留新 E-mail:quliuxin@aliyun.com
更新日期/Last Update: 1900-01-01