基于“调筋治骨”法探讨针刀治疗KOA模型兔软骨与股直肌修复的作用机制
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摘要
膝关节骨性关节炎(Knee Osteoarthritis,KOA)是40岁以上人群最常见的慢性、进展性、退行性关节疾病,它以关节软骨退变及骨质硬化、增生为主要特征,以进行性发展的膝关节疼痛、僵硬、肿胀、功能障碍为临床主要表现,严重时可导致关节畸形,甚至丧失关节功能,影响患者正常生活和工作。在全球范围内KOA约占成年人口的6%,发病率女性总体高于男性.约在1.5：1～4：1之间,该病有一定的致残率。但目前现代医学对其病因尚不完全清楚,也尚无根治本病的方案和预防措施。中医药在治疗KOA相关疾病疗效显著,尤其是针刀疗法在临床治疗KOA上无论是单独应用还是联合应用,均以其简、便、廉、验的优势成为治疗本病的主流疗法。目前,研究者们通过行为学、形态学、生物力学、分子生物学等方法和手段,从中枢镇痛、细胞因子、软骨细胞凋亡、细胞外基质等角度对针刀KOA治疗的作用机理开展了相关研究,而对针刀对于膝关节周围软组织的损伤与修复,以及骨骼肌损伤修复与软骨损伤修复之间关系的相关研究较少。本研究依托郭长青教授国家自然基金项目“针刀干预对膝关节炎韧带力学改变及软骨细胞力学信号转导的影响”的大量研究工作基础,以骨骼肌损伤修复及其与关节软骨损伤修复之间的关系为切入点,即从“筋”-“骨”平衡关系角度探讨针刀治疗KOA的疗效和机制。
目的：本实验以骨骼肌损伤修复及其与关节软骨的损伤修复之间关系为切入点,旨在验证针刀松解法通过“调筋治骨”恢复膝关节“筋骨平衡”思路的科学性,为以中医筋经理论指导下开展的KOA相关疗法提供依据。
方法：将6月龄,健康清洁级,新西兰兔45只,按随机原则分为5组：正常组、模型组、针刀组、电针组和圆利针组,每组9只,其中8只用于各项指标检测,1只用于造模后6周形态学检测造模成功。以改良后的Videman法左后肢伸直位固定制动法造模,对实验性KOA模型兔分别采用针刀、电针和圆利针三种针刺方法进行干预治疗,针刀和圆利针选取左侧内、外侧副韧带及髌韧带的中点前缘、起、止点(阿是穴)进入,刀口线与韧带方向平行,分别松解5次,每周1次,共6次；圆利针进针点与针刀组一致,分别松解5次,每周1次,共6次；电针选取“阳陵泉-阴陵泉”、“内膝眼-外膝眼”疏密波,连续波,频率2/100Hz,强度3mA,每次20min,隔日1次,1周3次,共6周对照。实验第一部分和第二部分通过观察不同时间窗下,KOA模型兔体重、兔膝关节屈伸被动活动范围(PROM), Lequesne MG的膝关节级别评估(关节局部反应、步态分析、关节畸形)等方面进行行为学观察,并通过X-ray, MRI对KOA兔膝关节系统整体情况进行影像学观察；同时通过HE染色在普通光学显微镜下观察实验性KOA兔股直肌细胞的形态、肌纤维断裂情况和异常细胞情况,以及观察关节软骨潮线及间隙等病理变化情况；并于透射电镜下观察股直肌超微结构变化,于扫描电镜下观察关节软骨表面超微结构变化,比较针刀、电针和圆利针三种干预治疗方法之间的疗效差异；实验第三部分和第四部分以TUNEL法检测KOA模型兔股直肌细胞凋亡AI指数,采用Western blot法检测股直肌中Bcl-2, Bax, Caspase-3的蛋白表达,采用Real-Time PCR和western blot检测病变关节软骨中MMP-1、3、13和TIMP-1mRNA和蛋白表达水平,探讨三种干预方法治疗实验性KOA对软组织损伤修复和软骨损伤修复之间关系的影响。
结果：
1、针刀、电针和圆利针三种干预治疗措施能够有效改善KOA模型兔关节疼痛和运动功能障碍,即Lequesne MG的膝关节炎总指数(疼痛、关节肿胀、关节畸形)与膝关节被动活动度(PROM)等行为学指标,同时对膝关节X-ray表现的关节面软骨破坏、关节间隙狭窄和骨赘形成均有明显的改善作用；对膝关节MRI表现滑膜增生、关节畸形也有一定的改善作用,且三个治疗方法的治疗效果无显著差异。
2、实验性KOA模型兔膝关节周围软组织与软骨损伤与修复同时存在,从形态学角度观察,针刀、电针和圆利针对于骨骼肌和软骨均有明确的修复作用。在光镜下,股直肌HE染色切片显示三种干预治疗方法均可有效抑制关节局部炎症反应,促进肌纤维结构恢复正常,且针刀组优于电针组和圆利针组,电针与圆利针组无显著差异；透射电镜下可见,针刀组、电针组和圆利针组骨骼肌纤维较模型组恢复了M线、Z线的有序排列,且可见大量线粒体聚集,部分新生肌丝生成等再生修复迹象,三种干预治疗效果无显著差异。关节软骨HE染色切片显示,针刀、电针和圆利针有效抑制了软骨基质的退变,促进了软骨的再生修复,且针刀组效果优于电针组和圆利针组,圆利针组优于电针组；扫描电镜下,可见三种疗法均有效改善了软骨表面结构的破坏,且针刀组优于电针组和圆利针组,圆利针组优于电针组。
3、从TUNEL结果显示中我们得出三种干预治疗方法降低了骨骼肌细胞凋亡,针刀组和圆利针组优于电针组(P<0.05); Bax蛋白表达：模型组、电针组较正常组显著升高(P<0.05)；与模型组比较,针刀组与圆利针组蛋白表达均明显降低(P<0.05),Bc1-2/Bax比值：模型组、电针组较正常组显著降低(P<0.05)；针刀组与圆利针组较模型组蛋白表达明显升高(P<0.05). Caspase-3蛋白表达：模型组与电针组较空白组明显升高(P<0.05),针刀组与圆利针组较模型组明显降低(P<0.05)。以松解手法为主的针刀和圆利针疗法可通过下调Bax、 Caspase-3蛋白表达,升高Bc1-2/Bax比值抑制KOA模型兔股直肌细胞凋亡,表明其治疗KOA的机制可能与抑制病变关节周围骨骼肌以线粒体途径为主细胞凋亡有关,这与电针治疗关节损伤的机制存在差异。
4. Real-Time PCR结果显示：正常组、模型组、针刀组、电针组和圆利针组各组间比较,MMP-1的mRNA表达无统计学差异(P>0.05); TIMP-1的mRNA表达各组较正常组有升高(P<0.05),电针组较模型组显著升高(P<0.05); MMP-3的mRNA表达,模型组、电针组与圆利针组较正常组显著升高(p<0.05),针刀组、电针组与圆利针组较模型组显著降低(p<0.05); MMP-13的mRNA表达模型组、电针组较正常组显著升高(p<0.05),针刀组与圆利针组较模型组显著降低(p<0.05)。Western blot实验结果显示：MMP-1蛋白表达水平模型组较正常组明显升高(P<0.01); TIMP-1蛋白表达模型组较正常组显著降低(P<0.05),电针组、针刀组与圆利针组较模型组显著升高(p<0.05); MMP-3. MMP-13蛋白表达模型组和电针组较正常组显著升高(p<0.05),针刀组与圆利针较模型组显著降低p<0.05)。以松解手法为主的针刀和圆利针对MMP-3. MMP-13的mRNA和蛋白表达有抑制作用,表明其治疗KOA的机制可能与调节基质金属蛋白酶家族有关,这与电针治疗关节损伤的机制存在差异。
结论：
1、针刀松解法可抑制膝关节局部炎症、滑膜增生等反应,降低软骨基质退变及骨质硬化、增生等现象,从而改善实验性KOA膝关节疼痛、肿胀及功能障碍。
2、针刀松解法能有效治疗实验性KOA模型兔股直肌和软骨组织的损伤,并能促进股直肌纤维和软骨细胞的再生修复,进而恢复组织的正常结构和功能。
3、针刀松解法可能通过抑制股直肌细胞凋亡,改善软骨细胞外基质MMPs之间的平衡,促进股直肌和软骨的同步修复,且其与电针治疗KOA的机制存在差异。
4、通过松解膝关节周围的软组织,解除关节内异常应力集中点,恢复关节的力学平衡,可能是针刀松解法治疗KOA的重要机制之一,即通过“调筋治骨”恢复“筋骨平衡”而达到治疗效果。
Background:Knee osteoarthritis (KOA) is the most common chronic, progressive, degenerative joint disease among patients over40years old. It is characterized by degeneration of articular cartilage, bone sclerosis, and hyperplasia manifested as progressive knee joint pain, stiffness, swelling, dysfunction of the knee joints, which may also develop into joint deformity, even loss of joint function, greatly affecting patients' normal life and work. Globally, KOA with high rate of disability, accounts for about6%of the adult population. Its incidence in women was slightly higher than that in men at around about1.5:1~4:1. However, its etiology and pathogenesis are still unclear. There is still no cure for the disease and preventive measures.
Traditional Chinese medicine has been shown to be effective for KOA, especially the acupotomy therapy in the treatment of KOA either alone or combined with other therapies. As it is easy, convenient, inexpensive and effective, it has become a mainstream therapy for this disease. At present, behavior, morphology, biomechanics, molecular biology approaches have been utilized in exploring the mechanism of acupotomy in the treatment KOA from perspectives of central analgesia, cytokine, cartilage cell apoptosis, and extracellular matrix. Studies on effects of the acupotomy therapy on soft tissue around the knee, and relation between skeletal muscle injury and articular cartilage repair have been rare.
This study attempts to explore the effect and mechanism of acupotomy for KOA based on a National Natural Science Foundation Project'Intervention effect of Acupotomy on KOA ligament mechanical changes and cartilage cell signal conduction' lead by Prof. Guo Changqing. The relationship between injury-repair of skeletal muscle and articular cartilage, or the sinew-bone balance, is the breakthrough point.
Objective:To verify the theory that modulating sinew (Jin) and treating bone (Gu) could resume their balance and achieve therapeutic effects, to provide the scientific basis for a variety of therapies in traditional Chinese medicine for KOA under the guidance of the theory of muscle-sinew theory.
Methods:Forty-five6-months old, health and sanitary leve, New Zealand rabbits were randomly divided into five groups (n=9in each group):normal control group, model group, acupotomy (AP) group, electro-acupuncture (EA) group and Round-sharp Acupuncture (RSA) group.8rabbits of each group were measured while the remaining one was used for validation of morphological modeling after4weeks. The left knee joint was fixed with plaster bandage in extension position to establish the rabbit model of knee joint injury with the Modified Videman method. Acupotomy, electro-acupuncture and round-sharp Acupuncture were respectively applied in AP, EA and RSA group.
EA group:left "Yanglingquan (GB34) with Yinlingquan (SP9)","Neixiyan (Ex-LE04) with Waixiyan (ST35)", using rarefaction-dense and continuous wave of2/100Hz,3mA, for20min each time,3times/week,3weeks in total;
AP group:the front edge of the midpoint, the starting point and the stopping point of the medial collateral ligament, lateral collateral ligament and patellar ligament. The ligament edge line and loosening direction should keep parallel,5times respectively, once a week,3weeks in total; the needle points of RAS group are same with AP group,5times respectively, once a week,3weeks in total.
In the first part and the second part of the study, bilateral knee passive range (PROM) and the Lequesne MG total index was evaluated respectively at different time window after modeling. X-ray and MRI were used to assess the radiological changes of KOA rabbit knee joints. HE staining was used to observe the morphology of rectus femoris muscle, myocardial fiber rupture and abnormal cells though the ordinary optical microscope. Tidal line clearance, articular cartilage and calcified layer and deep cartilage changes were measured. Ultra structural changes of the rectus femoris were observed using transmission electron microscope while those of articular cartilage surface, by scanning electron microscope, to compare the differences between the AP, EA and RSA therapy. In the third part and the fourth part, Apoptotic index was measured using TUNEL method. Expression of Bcl-2and baxand Caspase-3proteins were assessed with western blot tests. MMP-1, so were MMP-3, MMP-13, TIMP-1mRNA by RT-PCR and TIMP-1proteins by western blot tests.
Result:
1. AP, EA and RSA therapy can effectively relieve articular pain and movement disorder in KOA rabbits, by reducing Lequesne MG knee arthritis index (pain, joint swelling, and deformity of knee joint) and passive motion (PROM) index behavior significantly. X-ray findings of articular cartilage of knee joint damage, joint space narrowing and osteophytosis demonstrated significant improvement as well. It was also found to have a certain improving effect on knee joint MRI synovial hyperplasia, and joint deformities. The effects were similar among three groups.
2. Soft tissue and cartilage injury and repair appeared to exist at the same time around the knee joints in experimental KOA rabbits. From morphology observation, repairing effects of AP, EA and RSA therapy on skeletal muscle and cartilage were definite. Under the microscope, HE staining of the rectus femoris showed that all of the three therapies could effectively inhibit joint local inflammatory response, promoting the recovery of normal muscle fiber structure. AP group demonstrated improved effectiveness than EA and RSA group, while the latter two were similar in effects. Under transmission electron microscope, AP group, skeletal muscle fibers arrangement in EA and RSA groups appeared more orderly than those in the model group, as the M line, Z line was restored, the amount of mitochondrial bodies gathered, and presence of some of the new filament generation regeneration. The effects were similar among three groups. HE of the articular cartilage stained sections revealed that AP, EA and RSA therapy could effectively inhibit the cartilage degeneration, promote regeneration and repair of cartilage with AP group being superior to EA and RSA group, RSA group better than EA group. Under the scanning electron microscope, it seemed that three therapies could all effectively improve the surface structure of cartilage destruction with AP group being superior to EA and RSA group, and RSA group better than EA group.
3. TUNEL findings showed that among the three types of intervention methods for reducing the apoptosis of skeletal muscle cells, AP group and RSA group were more effective than EA group (P0.05); Expression of Bax and Caspase-3proteins, model group and EA group increased significantly (P <0.05) compared with normal control group; AP group and RSA group decreased significantly (P <0.05) compared with model group, while the results were opposite in terms of the ratio of Bcl-2/Bax. AP and RSA therapy using relaxing methods could reduce the Bax, Caspase-3protein expression, increase the ratio of Bcl-2/Bax, and inhibit the apoptosis of the rectus femoris in KOA rabbits. This showed that the treatment of KOA mechanism may be related to the inhibition of skeletal muscle lesions around the joint, mainly through the mitochondria pathway of apoptosis, the mechanism of which seemed different from E A.
4. Low level expressions of MMP-1mRNA were observed in cartilage damage at4weeks after modeling, and there was no significant difference between groups (p>0.05). Expression of MMP-1protein decreased significantly (P<0.05) in model group compared with normal control group. In comparison with normal control group after modeling, TIMP-1mRNA increased significantly (P<0.05) in model, EA, AP and RSA groups, and it was much higher in EA group than that in model group (P<0.05). Expression of TIMP-1protein decreased significantly (P<0.05) in model and AP group when compared with normal control group. But it increased significantly in EA and RSA group compared with model group (P<0.05). Expression of MMP3, MMP13mRNA and proteins increased significantly (P<0.05) in EA group and model group, in comparison with normal control group while the expressions in AP group and RSA group decreased significantly (P<0.05), and MMP3mRNA level in EA and model group was much higher than that in normal control group (P <0.05) and lower in AP and RSA group compared with model group (P<0.05). AP and RSA therapy seemed to have inhibitive effects on MMP-3, MMP-13mRNA and protein expression in the articular cartilage of KOA rabbits. It suggests that the mechanism of AP and RSA therapy may be related to regulation of matrix metalloproteinase family, which is different from that of EA.
Conclusion:
1. Acupotomy therapy can inhibit the knee joint inflammation, synovial proliferation reaction, and reduce the degeneration of cartilage matrix and bone sclerosis, hyperplasia and other pathology, so as to improve the KOA knee joint pain, swelling and dysfunction
2. Acupotomy therapy can improve the injury of the femoral rectus and cartilage tissue of KOA effectively, and can promote the regeneration and repair of muscle fiber and cartilage cells; the normal structure and function would be thus restored
3. Acupotomy therapy may inhibit the rectus femoris muscle cell apoptosis, improve the extracellular matrix MMPs balance of cartilage, and promote the repair of rectus femoris and cartilage. This mechanism is different from EA.
4. AP therapy probably works mainly by releasing the soft tissue around knew joints and relieving tension within the joint to restore the mechanical balance of the joint in the treatment of KOA. It is so-called modulating sinews and treating bones to achieve therapeutic effects.
目的：本实验以骨骼肌损伤修复及其与关节软骨的损伤修复之间关系为切入点,旨在验证针刀松解法通过“调筋治骨”恢复膝关节“筋骨平衡”思路的科学性,为以中医筋经理论指导下开展的KOA相关疗法提供依据。
方法：将6月龄,健康清洁级,新西兰兔45只,按随机原则分为5组：正常组、模型组、针刀组、电针组和圆利针组,每组9只,其中8只用于各项指标检测,1只用于造模后6周形态学检测造模成功。以改良后的Videman法左后肢伸直位固定制动法造模,对实验性KOA模型兔分别采用针刀、电针和圆利针三种针刺方法进行干预治疗,针刀和圆利针选取左侧内、外侧副韧带及髌韧带的中点前缘、起、止点(阿是穴)进入,刀口线与韧带方向平行,分别松解5次,每周1次,共6次；圆利针进针点与针刀组一致,分别松解5次,每周1次,共6次；电针选取“阳陵泉-阴陵泉”、“内膝眼-外膝眼”疏密波,连续波,频率2/100Hz,强度3mA,每次20min,隔日1次,1周3次,共6周对照。实验第一部分和第二部分通过观察不同时间窗下,KOA模型兔体重、兔膝关节屈伸被动活动范围(PROM), Lequesne MG的膝关节级别评估(关节局部反应、步态分析、关节畸形)等方面进行行为学观察,并通过X-ray, MRI对KOA兔膝关节系统整体情况进行影像学观察；同时通过HE染色在普通光学显微镜下观察实验性KOA兔股直肌细胞的形态、肌纤维断裂情况和异常细胞情况,以及观察关节软骨潮线及间隙等病理变化情况；并于透射电镜下观察股直肌超微结构变化,于扫描电镜下观察关节软骨表面超微结构变化,比较针刀、电针和圆利针三种干预治疗方法之间的疗效差异；实验第三部分和第四部分以TUNEL法检测KOA模型兔股直肌细胞凋亡AI指数,采用Western blot法检测股直肌中Bcl-2, Bax, Caspase-3的蛋白表达,采用Real-Time PCR和western blot检测病变关节软骨中MMP-1、3、13和TIMP-1mRNA和蛋白表达水平,探讨三种干预方法治疗实验性KOA对软组织损伤修复和软骨损伤修复之间关系的影响。
结果：
1、针刀、电针和圆利针三种干预治疗措施能够有效改善KOA模型兔关节疼痛和运动功能障碍,即Lequesne MG的膝关节炎总指数(疼痛、关节肿胀、关节畸形)与膝关节被动活动度(PROM)等行为学指标,同时对膝关节X-ray表现的关节面软骨破坏、关节间隙狭窄和骨赘形成均有明显的改善作用；对膝关节MRI表现滑膜增生、关节畸形也有一定的改善作用,且三个治疗方法的治疗效果无显著差异。
2、实验性KOA模型兔膝关节周围软组织与软骨损伤与修复同时存在,从形态学角度观察,针刀、电针和圆利针对于骨骼肌和软骨均有明确的修复作用。在光镜下,股直肌HE染色切片显示三种干预治疗方法均可有效抑制关节局部炎症反应,促进肌纤维结构恢复正常,且针刀组优于电针组和圆利针组,电针与圆利针组无显著差异；透射电镜下可见,针刀组、电针组和圆利针组骨骼肌纤维较模型组恢复了M线、Z线的有序排列,且可见大量线粒体聚集,部分新生肌丝生成等再生修复迹象,三种干预治疗效果无显著差异。关节软骨HE染色切片显示,针刀、电针和圆利针有效抑制了软骨基质的退变,促进了软骨的再生修复,且针刀组效果优于电针组和圆利针组,圆利针组优于电针组；扫描电镜下,可见三种疗法均有效改善了软骨表面结构的破坏,且针刀组优于电针组和圆利针组,圆利针组优于电针组。
3、从TUNEL结果显示中我们得出三种干预治疗方法降低了骨骼肌细胞凋亡,针刀组和圆利针组优于电针组(P<0.05); Bax蛋白表达：模型组、电针组较正常组显著升高(P<0.05)；与模型组比较,针刀组与圆利针组蛋白表达均明显降低(P<0.05),Bc1-2/Bax比值：模型组、电针组较正常组显著降低(P<0.05)；针刀组与圆利针组较模型组蛋白表达明显升高(P<0.05). Caspase-3蛋白表达：模型组与电针组较空白组明显升高(P<0.05),针刀组与圆利针组较模型组明显降低(P<0.05)。以松解手法为主的针刀和圆利针疗法可通过下调Bax、 Caspase-3蛋白表达,升高Bc1-2/Bax比值抑制KOA模型兔股直肌细胞凋亡,表明其治疗KOA的机制可能与抑制病变关节周围骨骼肌以线粒体途径为主细胞凋亡有关,这与电针治疗关节损伤的机制存在差异。
4. Real-Time PCR结果显示：正常组、模型组、针刀组、电针组和圆利针组各组间比较,MMP-1的mRNA表达无统计学差异(P>0.05); TIMP-1的mRNA表达各组较正常组有升高(P<0.05),电针组较模型组显著升高(P<0.05); MMP-3的mRNA表达,模型组、电针组与圆利针组较正常组显著升高(p<0.05),针刀组、电针组与圆利针组较模型组显著降低(p<0.05); MMP-13的mRNA表达模型组、电针组较正常组显著升高(p<0.05),针刀组与圆利针组较模型组显著降低(p<0.05)。Western blot实验结果显示：MMP-1蛋白表达水平模型组较正常组明显升高(P<0.01); TIMP-1蛋白表达模型组较正常组显著降低(P<0.05),电针组、针刀组与圆利针组较模型组显著升高(p<0.05); MMP-3. MMP-13蛋白表达模型组和电针组较正常组显著升高(p<0.05),针刀组与圆利针较模型组显著降低p<0.05)。以松解手法为主的针刀和圆利针对MMP-3. MMP-13的mRNA和蛋白表达有抑制作用,表明其治疗KOA的机制可能与调节基质金属蛋白酶家族有关,这与电针治疗关节损伤的机制存在差异。
结论：
1、针刀松解法可抑制膝关节局部炎症、滑膜增生等反应,降低软骨基质退变及骨质硬化、增生等现象,从而改善实验性KOA膝关节疼痛、肿胀及功能障碍。
2、针刀松解法能有效治疗实验性KOA模型兔股直肌和软骨组织的损伤,并能促进股直肌纤维和软骨细胞的再生修复,进而恢复组织的正常结构和功能。
3、针刀松解法可能通过抑制股直肌细胞凋亡,改善软骨细胞外基质MMPs之间的平衡,促进股直肌和软骨的同步修复,且其与电针治疗KOA的机制存在差异。
4、通过松解膝关节周围的软组织,解除关节内异常应力集中点,恢复关节的力学平衡,可能是针刀松解法治疗KOA的重要机制之一,即通过“调筋治骨”恢复“筋骨平衡”而达到治疗效果。
Background:Knee osteoarthritis (KOA) is the most common chronic, progressive, degenerative joint disease among patients over40years old. It is characterized by degeneration of articular cartilage, bone sclerosis, and hyperplasia manifested as progressive knee joint pain, stiffness, swelling, dysfunction of the knee joints, which may also develop into joint deformity, even loss of joint function, greatly affecting patients' normal life and work. Globally, KOA with high rate of disability, accounts for about6%of the adult population. Its incidence in women was slightly higher than that in men at around about1.5:1~4:1. However, its etiology and pathogenesis are still unclear. There is still no cure for the disease and preventive measures.
Traditional Chinese medicine has been shown to be effective for KOA, especially the acupotomy therapy in the treatment of KOA either alone or combined with other therapies. As it is easy, convenient, inexpensive and effective, it has become a mainstream therapy for this disease. At present, behavior, morphology, biomechanics, molecular biology approaches have been utilized in exploring the mechanism of acupotomy in the treatment KOA from perspectives of central analgesia, cytokine, cartilage cell apoptosis, and extracellular matrix. Studies on effects of the acupotomy therapy on soft tissue around the knee, and relation between skeletal muscle injury and articular cartilage repair have been rare.
This study attempts to explore the effect and mechanism of acupotomy for KOA based on a National Natural Science Foundation Project'Intervention effect of Acupotomy on KOA ligament mechanical changes and cartilage cell signal conduction' lead by Prof. Guo Changqing. The relationship between injury-repair of skeletal muscle and articular cartilage, or the sinew-bone balance, is the breakthrough point.
Objective:To verify the theory that modulating sinew (Jin) and treating bone (Gu) could resume their balance and achieve therapeutic effects, to provide the scientific basis for a variety of therapies in traditional Chinese medicine for KOA under the guidance of the theory of muscle-sinew theory.
Methods:Forty-five6-months old, health and sanitary leve, New Zealand rabbits were randomly divided into five groups (n=9in each group):normal control group, model group, acupotomy (AP) group, electro-acupuncture (EA) group and Round-sharp Acupuncture (RSA) group.8rabbits of each group were measured while the remaining one was used for validation of morphological modeling after4weeks. The left knee joint was fixed with plaster bandage in extension position to establish the rabbit model of knee joint injury with the Modified Videman method. Acupotomy, electro-acupuncture and round-sharp Acupuncture were respectively applied in AP, EA and RSA group.
EA group:left "Yanglingquan (GB34) with Yinlingquan (SP9)","Neixiyan (Ex-LE04) with Waixiyan (ST35)", using rarefaction-dense and continuous wave of2/100Hz,3mA, for20min each time,3times/week,3weeks in total;
AP group:the front edge of the midpoint, the starting point and the stopping point of the medial collateral ligament, lateral collateral ligament and patellar ligament. The ligament edge line and loosening direction should keep parallel,5times respectively, once a week,3weeks in total; the needle points of RAS group are same with AP group,5times respectively, once a week,3weeks in total.
In the first part and the second part of the study, bilateral knee passive range (PROM) and the Lequesne MG total index was evaluated respectively at different time window after modeling. X-ray and MRI were used to assess the radiological changes of KOA rabbit knee joints. HE staining was used to observe the morphology of rectus femoris muscle, myocardial fiber rupture and abnormal cells though the ordinary optical microscope. Tidal line clearance, articular cartilage and calcified layer and deep cartilage changes were measured. Ultra structural changes of the rectus femoris were observed using transmission electron microscope while those of articular cartilage surface, by scanning electron microscope, to compare the differences between the AP, EA and RSA therapy. In the third part and the fourth part, Apoptotic index was measured using TUNEL method. Expression of Bcl-2and baxand Caspase-3proteins were assessed with western blot tests. MMP-1, so were MMP-3, MMP-13, TIMP-1mRNA by RT-PCR and TIMP-1proteins by western blot tests.
Result:
1. AP, EA and RSA therapy can effectively relieve articular pain and movement disorder in KOA rabbits, by reducing Lequesne MG knee arthritis index (pain, joint swelling, and deformity of knee joint) and passive motion (PROM) index behavior significantly. X-ray findings of articular cartilage of knee joint damage, joint space narrowing and osteophytosis demonstrated significant improvement as well. It was also found to have a certain improving effect on knee joint MRI synovial hyperplasia, and joint deformities. The effects were similar among three groups.
2. Soft tissue and cartilage injury and repair appeared to exist at the same time around the knee joints in experimental KOA rabbits. From morphology observation, repairing effects of AP, EA and RSA therapy on skeletal muscle and cartilage were definite. Under the microscope, HE staining of the rectus femoris showed that all of the three therapies could effectively inhibit joint local inflammatory response, promoting the recovery of normal muscle fiber structure. AP group demonstrated improved effectiveness than EA and RSA group, while the latter two were similar in effects. Under transmission electron microscope, AP group, skeletal muscle fibers arrangement in EA and RSA groups appeared more orderly than those in the model group, as the M line, Z line was restored, the amount of mitochondrial bodies gathered, and presence of some of the new filament generation regeneration. The effects were similar among three groups. HE of the articular cartilage stained sections revealed that AP, EA and RSA therapy could effectively inhibit the cartilage degeneration, promote regeneration and repair of cartilage with AP group being superior to EA and RSA group, RSA group better than EA group. Under the scanning electron microscope, it seemed that three therapies could all effectively improve the surface structure of cartilage destruction with AP group being superior to EA and RSA group, and RSA group better than EA group.
3. TUNEL findings showed that among the three types of intervention methods for reducing the apoptosis of skeletal muscle cells, AP group and RSA group were more effective than EA group (P
4. Low level expressions of MMP-1mRNA were observed in cartilage damage at4weeks after modeling, and there was no significant difference between groups (p>0.05). Expression of MMP-1protein decreased significantly (P<0.05) in model group compared with normal control group. In comparison with normal control group after modeling, TIMP-1mRNA increased significantly (P<0.05) in model, EA, AP and RSA groups, and it was much higher in EA group than that in model group (P<0.05). Expression of TIMP-1protein decreased significantly (P<0.05) in model and AP group when compared with normal control group. But it increased significantly in EA and RSA group compared with model group (P<0.05). Expression of MMP3, MMP13mRNA and proteins increased significantly (P<0.05) in EA group and model group, in comparison with normal control group while the expressions in AP group and RSA group decreased significantly (P<0.05), and MMP3mRNA level in EA and model group was much higher than that in normal control group (P <0.05) and lower in AP and RSA group compared with model group (P<0.05). AP and RSA therapy seemed to have inhibitive effects on MMP-3, MMP-13mRNA and protein expression in the articular cartilage of KOA rabbits. It suggests that the mechanism of AP and RSA therapy may be related to regulation of matrix metalloproteinase family, which is different from that of EA.
Conclusion:
1. Acupotomy therapy can inhibit the knee joint inflammation, synovial proliferation reaction, and reduce the degeneration of cartilage matrix and bone sclerosis, hyperplasia and other pathology, so as to improve the KOA knee joint pain, swelling and dysfunction
2. Acupotomy therapy can improve the injury of the femoral rectus and cartilage tissue of KOA effectively, and can promote the regeneration and repair of muscle fiber and cartilage cells; the normal structure and function would be thus restored
3. Acupotomy therapy may inhibit the rectus femoris muscle cell apoptosis, improve the extracellular matrix MMPs balance of cartilage, and promote the repair of rectus femoris and cartilage. This mechanism is different from EA.
4. AP therapy probably works mainly by releasing the soft tissue around knew joints and relieving tension within the joint to restore the mechanical balance of the joint in the treatment of KOA. It is so-called modulating sinews and treating bones to achieve therapeutic effects.
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