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[1]張力,張華,李平,等.中醫(yī)整脊棘突旁指壓法干預大鼠膝骨關節(jié)炎的效果及作用機制研究[J].中醫(yī)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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中醫(yī)整脊棘突旁指壓法干預大鼠膝骨關節(jié)炎的效果及作用機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年03期
頁碼:
14-22,38
欄目:
基礎研究
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:
張力; 張華; 李平; 高嵩; 柯廣娟; 屈留新
東南大學附屬中大醫(yī)院,江蘇 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
關鍵詞:
骨關節(jié)炎; 膝;  中醫(yī)整脊;  指壓法;  疼痛;  敏化;  瞬時受體電位香草素受體1;  神經(jīng)節(jié); 脊;  神經(jīng)元;  滑膜;  成纖維細胞;  串話;  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:探討中醫(yī)整脊棘突旁指壓法干預大鼠膝骨關節(jié)炎(knee osteoarthritis,KOA)的效果及作用機制。方法:將40只SD大鼠隨機分為空白組、模型組、低強度組、中強度組和高強度組,每組8只。模型組、低強度組、中強度組和高強度組大鼠均采用碘乙酸鈉關節(jié)腔注射建立雙側KOA模型,空白組大鼠雙側膝關節(jié)腔注射等量生理鹽水。造模14 d后,采用小動物計力指壓器于L3～L5棘突旁,對低、中、高強度組大鼠分別施以4 N、8 N、16 N的垂直向下壓力,模擬不同力度的棘突旁指壓法; 每次按壓2 s,每處連續(xù)按壓2 min; 每隔1 d施術1次,干預28 d; 空白組和模型組不進行任何干預。分別于干預1 d、7 d、14 d、21 d、28 d,采用von Frey電子測痛儀測定大鼠機械刺激縮足閾值(paw withdrawal mechanical threshold,PWMT)。干預28 d后,采用ELISA試劑盒檢測大鼠血清中神經(jīng)生長因子(nerve growth factor,NGF)、降鈣素基因相關肽(calcitonin gene related peptide,CGRP)含量; 采用鈣成像技術檢測大鼠L3背根神經(jīng)節(jié)神經(jīng)元Ca2+通量; 采用Western blot法檢測大鼠L3背根神經(jīng)節(jié)組織中瞬時受體電位香草素受體1(transient receptor potential vanilloid 1,TRPV1)、脂蛋白磷酸酶2B(protein phosphatases 2B,PP2B)、鈣調蛋白(calmodulin,CaM)的蛋白表達水平; 分別采用HE染色試劑盒、Masson染色試劑盒、天狼星紅染色試劑盒對大鼠膝關節(jié)滑膜組織進行染色,觀察滑膜組織的炎性細胞浸潤和纖維化情況; 采用Western blot法檢測大鼠膝關節(jié)滑膜組織中白細胞介素(interleukin,IL)-1β、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、轉化生長因子(transforming growth factor,TGF)-β、α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)的蛋白表達水平。結果:①PWMT測定結果。空白組、模型組、低強度組大鼠PWMT隨時間變化均基本保持不變(F=0.885,P=0.487; F=0.339,P=0.849; F=0.555,P=0.697),中、高強度組大鼠PWMT隨時間變化均呈上升趨勢(F=8.222,P=0.000; F=9.844,P=0.000)。干預21 d、28 d,中、高強度組大鼠PWMT均高于模型組(干預21 d:P=0.002,P=0.001; 干預 28 d:P=0.003,P=0.000)和低強度組(干預21 d:P=0.022,P=0.011; 干預28 d:P=0.044,P=0.003),高強度組大鼠PWMT與中強度組比較,差異均無統(tǒng)計學意義(P=0.774,P=0.273)。②血清疼痛介質含量測定結果。中、高強度組大鼠血清NGF、CGRP含量均低于模型組(NGF含量:P=0.003,P=0.000; CGRP含量:P=0.000,P=0.000); 中強度組大鼠血清NGF含量與低強度組比較,差異無統(tǒng)計學意義(P=1.000),血清CGRP含量低于低強度組(P=0.000); 高強度組大鼠血清NGF、CGRP含量均低于低強度組(P=0.027,P=0.000); 高強度組大鼠血清NGF、CGRP含量與中強度組比較,組間差異均無統(tǒng)計學意義(P=1.000,P=1.000)。③L3背根神經(jīng)節(jié)神經(jīng)元Ca2+通量檢測結果。低、中、高強度組大鼠L3背根神經(jīng)節(jié)神經(jīng)元Ca2+平均熒光強度均高于模型組(P=0.001,P=0.000,P=0.000),中、高強度組大鼠L3背根神經(jīng)節(jié)神經(jīng)元Ca2+平均熒光強度均高于低強度組(P=0.003,P=0.016),高強度組大鼠L3背根神經(jīng)節(jié)神經(jīng)元Ca2+平均熒光強度與中強度組比較,差異無統(tǒng)計學意義(P=0.361)。④L3背根神經(jīng)節(jié)組織中TRPV1、PP2B、CaM的蛋白表達水平檢測結果。中、高強度組大鼠L3背根神經(jīng)節(jié)組織中TRPV1的蛋白相對表達量均高于模型組和低強度組(模型組:P=0.002,P=0.000; 低強度組:P=0.047,P=0.014),PP2B、CaM的蛋白相對表達量均低于模型組和低強度組(模型組:P=0.011,P=0.000; P=0.000,P=0.000; 低強度組:P=0.043,P=0.033; P=0.000,P=0.000); 高強度組大鼠L3背根神經(jīng)節(jié)組織中TRPV1、PP2B、CaM的蛋白相對表達量與中強度組比較,組間差異均無統(tǒng)計學意義(P=1.000,P=1.000,P=1.000)。⑤膝關節(jié)滑膜組織病理學觀察結果。HE染色顯示,模型組大鼠滑膜組織中呈現(xiàn)明顯的炎性細胞浸潤,低、中、高強度組大鼠滑膜組織中炎性細胞浸潤較模型組減輕,且中、高強度組減輕明顯; Masson染色顯示,模型組大鼠滑膜組織中膠原沉積較空白組明顯增加,低、中、高強度組大鼠滑膜組織中膠原沉積較模型組減少,且中、高強度組減少明顯; 天狼星紅染色顯示,模型組大鼠滑膜組織中Ⅱ型膠原明顯減少,低、中、高強度組大鼠滑膜組織中Ⅱ型膠原較模型組增多,且中、高強度組增多明顯。⑥膝關節(jié)滑膜組織中IL-1β、TNF-α、TGF-β、α-SMA的蛋白表達水平檢測結果。中、高強度組大鼠滑膜組織中IL-1β、TNF-α、TGF-β、α-SMA的蛋白相對表達量均低于模型組和低強度組(模型組:P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000; 低強度組:P=0.004,P=0.002,P=0.003,P=0.000; P=0.005,P=0.008,P=0.007,P=0.004); 高強度組大鼠滑膜組織中IL-1β、TNF-α、TGF-β、α-SMA的蛋白相對表達量與中強度組比較,組間差異均無統(tǒng)計學意義(P=0.116,P=0.083,P=0.224,P=0.341)。結論:中醫(yī)整脊棘突旁指壓法干預大鼠KOA,能夠減輕機械性疼痛敏化和滑膜炎癥,且作用效果與力度有關; 其作用機制可能與其能引起背根神經(jīng)節(jié)神經(jīng)元TRPV1脫敏及影響背根神經(jīng)節(jié)神經(jīng)元與滑膜成纖維細胞間串話有關。
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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備注/Memo

備注/Memo:
基金項目:東南大學附屬中大醫(yī)院中西醫(yī)協(xié)同醫(yī)學科研項目(2023zxyxt03)
通訊作者:屈留新 E-mail:[email protected]
更新日期/Last Update: 1900-01-01