针刀干预对膝骨关节炎兔股直肌纤维化的影响

Effect of Acupotomy Intervention on Fibrosis of Rectus Femoris in Rabbits with Knee Osteoarthritis

目的:从组织学、分子学层面分别观察针刀干预对膝骨关节炎兔股直肌纤维化的影响,揭示针刀治疗膝骨关节炎可能的作用机制。方法:选取体质量(2.0±0.5)kg的24只健康雄性新西兰兔,采用随机数字表法随机分为空白组、模型组和针刀组,每组8只。采用改良Videman法左后肢伸直位石膏固定制动6周复制KOA兔模型,参照Lequesne MG膝关节级别评分、X线和MRI膝关节影像学检测及股直肌病灶点触诊评价造模成功。针刀组采用经筋理论指导下在股四头肌常见经筋病灶点"鹤顶次"(足阳明经筋病灶点,股四头肌肌腱髌骨正上缘附着处)"、髌外上"(足阳明经筋病灶点,股外侧肌肌腱髌骨外上缘附着处)"、髌内上"(足阳明经筋病灶点,股内侧肌肌腱髌骨内上缘附着处)上行针刀松解治疗,每周1次,共治疗4次。空白组和模型组只做同样的抓取和固定,不予针刀治疗。干预结束1周后,采用Masson染色检测股直肌组织形态和胶原容积分数改变观察针刀对膝骨关节炎兔股直肌纤维化的影响,RT-PCR和Western blot检测股直肌Ⅰ型胶原纤维(ColⅠ)、波形蛋白(VIM)、α-肌动蛋白(α-SMA)mRNA和蛋白表达水平观察针刀对膝骨关节炎兔股直肌纤维化相关mRNA和蛋白的影响。结果:(1)Masson染色结果显示,空白组可见整齐的被红染的骨骼肌纤维;模型组可见骨骼肌纤维破坏,被大量蓝染的胶原纤维包绕;针刀组可见骨骼肌纤维较为整齐,被少量蓝染的胶原纤维包绕。模型组股直肌胶原容积分数较空白组增加,差异有统计学意义(P<0.01);针刀组较模型组股直肌胶原容积分数减小,差异有统计学意义(P<0.01)。(2)RT-PCR结果显示,模型组ColⅠ、VIM、α-SMA mRNA表达较空白组升高,差异有统计学意义(P<0.01);针刀组较模型组降低,差异有统计学意义(P<0.01)。(3)Western blot结果显示,模型组ColⅠ、VIM、α-SMA蛋白表达较空白组升高,差异有统计学意义(P<0.01);针刀组较模型组降低,差异有统计学意义(P<0.01)。结论:基于经筋理论针刀松解股四头肌经筋病灶点能下调ColⅠ、VIM、α-SMA mRNA和蛋白的表达水平,减轻股直肌纤维化程度,这可能是针刀治疗KOA作用机制之一。

Objective:To observe the effects of acupotomy intervention on the fibrosis of the rectus femoris in rabbits with knee osteoarthritis from the histological and molecular level,and to reveal the possible mechanism of the needle knife in the treatment of knee osteoarthritis.Methods:Twenty-four healthy male New Zealand rabbits were randomly divided into blank group,model group and acupotomy group used by random number table method,with 8 rabbits in each group.Modified Videman method was used to replicate the osteoarthritis model of the knee with left hind limb extended plaster immobility for 6 weeks.Lequesne MG knee joint grade score,X-ray and MRI imaging examination of knee joint and pathological morphology of rectus femoris were confirmed after successful modeling.In acupotomy group,the transfascial focal points of quadriceps femoris muscle were released by acupotomy under the guidance of Jingjin theory,treatment points included"Hedingci"(the focal lesion point of the foot yangming meridian,located at the attachment of the positive upper margin of the quadriceps tendon and the patella),"Binwaishang"(the focal lesion point of the foot yangming meridian,located at the attachment of the lateral upper edge of the patella to the lateral femoral muscle tendon),"Binneishang"(the focal lesion point of the foot yangming meridian,located at the attachment of the medial femoral muscle tendon to the inner upper edge of the patella),once a week,for a total of 4 times of treatment.The blank group and model group were routinely fed and grabbed without intervention.One week after the end of intervention,masson staining was used to detect the changes in the tissue morphology and collagen volume fraction of the rectus femoris to observe the effect of acupotomy on the fibrosis of the rectus femoris in rabbits with knee osteoarthritis.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of typeⅠcollagen fibers(ColⅠ),vimentin(VIM),and Alpha-smooth muscle actin(α-SMA)in the rectus femoris muscle to observe the effect of acupotomy on the knee osteoarthritis.Results:1)Masson staining results showed that neat red-stained skeletal muscle fibers can be seen in the blank group;skeletal muscle fibers can be seen in the model group damaged and surrounded by a large amount of blue-stained collagen fibers;in the acupotomy group,skeletal muscle fibers can be seen neatly and a small amount of blue-stained collagen fibers wrap around.Compared with the blank group,the collagen volume fraction of rectus femoris muscle in the model group increased,and the difference was statistically significant(P<0.01).Compared with the model group,the acupotomy group had decreased collagen volume fraction of rectus femoris muscle,and the difference was statistically significant(P<0.01).2)RT-PCR results showed that the mRNA expressions of ColⅠ,VIM andα-SMA in the model group were significantly higher than those in the blank group(P<0.01),while those in the acupotomy group were significantly lower than those in the model group,and the difference was statistically significant(P<0.01).3)Western blot results showed that the proteins expressions of ColⅠ,VIM andα-SMA in the model group were significantly higher than those in the blank group(P<0.01),while those in the acupotomy group were significantly lower than those in the model group,and the difference was statistically significant(P<0.01).Conclusion:Based on the Jingjin theory,the release of quadriceps femoris’transfascial focal point by acupotomy can down-regulate the expression levels of mRNA and protein of ColⅠ,VIM,α-SMA,and reduce the degree on fibrosis of rectus femoris muscle,which may be one of the mechanisms of acupotomy for KOA.