| Cervical spondylosis (CS) is becoming an increasingly common health problem throughout the world. It accounts for a large proportion in occupation-related diseases and disabilities. It causes a considerable impact and economic burden on individuals and their families, communities, health-care systems. The prevalence of CS has been increasing quickly, showing a trend of generalization and impacting the younger generation more. The clincal manifestations of CS are complicated and multilevel.It usually manifestates as pain on the neck and shoulder, headache, diziness, neck stiffness, numbness and weakness of upper limbs, reduction or loss of sensations. Prevention and treatment of CS has been a priority health problem, and study on CS has important significance in achieving this. As a new therapy of chinese medicine, acupotomy has been spreaded quickly since its advent in 1970s. Acupotomy has become a common therapy for CS. CS is a dominant disease indicating the value of necessitating acupotomy therapy. Acupotomy has achieved satisfactory efficacy, however its basic research on the efficacy mechanism is eagerly to be conducted.ObjectiveThis study established rabbit models of CS by the method of imitating human long-term flexed neck posture during work, a very common risk factor of human CS. Through observing the morphology of posterior cervical extensor muscles and intervertebral disc of rabbits with CS, changes of cervical X-rays taken before and after modeling, and after treatment, was used to assess the role of injury to the posterior cervical extensor muscles in the development of CS. Acupotomy therapy was applied after modeling, and compared to the group treated with electro-acupuncture. TUNEL-positive myonuclei count of posterior cervical extensor muscles, expression of apoptosis genes and proteins Bcl-2, Bax, Caspase-3 were tested. It is expected to explore the possible mechanism of acupotomy in the treatment of CS from the aspect of adjusting apoptosis of the posterior cervical extensor muscles and to provide evidences for acuptomy therapy.MethodsSix months old healthy adult New Zealand rabbits(with gender equality) were divided into four groups randomly:normal control group, model group, acupotomy group, and electro-acupuncture (EA) group. CS models were made by the method of flexed neck posture for long-term. After modeling, acupotomy and electro-acupuncture were applied to treat in acupotomy group and electro-acupuncture group. Both groups were treated for three weeks, once every week in acupotomy group, and three times in electro-acupuncture group. All the animals were subjected to cervical X-ray for three times:before and after modeling, and after treatment. Pathological changes of the muscles and intervertebral discs by hematoxylin-eosin staining were observed. TUNEL-positive myonuclei count of the muscles was conducted by TUNEL test. Expression of apoptosis genes Bcl-2 mRNA, Bax mRNA, Caspase-3 mRNA were conducted by Real-time PCR test, and expression of apoptosis proteins Bcl-2, Bax, Caspase-3 were conducted by Western blot test.Results1. Histomorphology of the muscle fibres in model group showed uneven staining, rhabdomylosis, increase of myocyte nuclears, nuclears moved to the middle of myocytes, fibrosis and scar formation, inflammatory cells aggregated, wave-shaped muscle fibres, lysis of muscle fibres, swollen muscle fibres, overgrowth of intercellular substance. In acupotomy group, histomorphology showed strgight-shaped muslcle fibres. Acupotomy promoted the repair of injuried muscle tissues. Histomorphology of intervertebral disc in model group showed nucleus pulposus was almosted replaced by fibers; overgrowth of collagenous fiber with a lot of chondrocytes-like cells were seen, and the direction of chondrocytes-like cells was consistent to collagenous fibers. Fibrosis of nucleus pulposus in acupotomy and electro-acupuncture groups was a little less than the model group and overgrowth of collagenous fibers was a little less than the model group.2. After modeling, cervical X-rays showed that there were changes in cervical lordosis, even lordotic curve with posterior convexity, a decrease in discs height, narrowed intervertebrale foramen and overgrowth of osteophytes in anterior and posterior vertebral bodies in some rabbits. There was obvious improvement in X-rays after acupotomy treatment; cervical lordosis was recovered to straight, intervertebral foramen widened in some rabbits, and there was no obvious change of osteophytes before and after treatment. After modeling, the cervical X-ray scores of the model group, acupotomy group and electro-acupuncture group increased significantly in comparison with normal control group (P<0.01). The reduction of X-ray scores before and after treatment in acupotomy group had a significant difference in comparison with model group (P<0.01).The reduction of X-ray scores before and after treatment in electro-acupuncture group had no significant difference with model group (P >0.05). The reduction of X-ray scores before and after treatment in acupotomy group had a significant difference in correlation with the electro-acupuncture group (P<0.05).3. In comparison with normal control group after modeling, TUNEL-positive myonuclei count of posterior cervical extensor muscles increased significantly (P<0.01). In comparison with normal control group, TUNEL-positive myonuclei count in acupotomy group increased mildly but there was no significant difference between the two groups (P>0.05).In comparison with model group, TUNEL-positive myonuclei count in acupotomy group decreased significantly (P<0.01). TUNEL-positive myonuclei count in electro-acupuncture group was higher than normal control group (P<0.01). In comparison with model group, TUNEL-positive myonuclei count in electro-acupuncture group decreased mildly, but there was no significant difference between the two groups (P>0.05). TUNEL-positive myonuclei count in acupotomy group was significantly lower than electro-acupuncture group (P<0.01).4. The results of Real-time PCR tests showed that there was no significant difference of expression of Bcl-2 mRNA between all the groups (P>0.05). In comparison with normal control group after modeling, Bax mRNA and Caspase-3 mRNA expression increased significantly (P<0.01, P<0.05), and the ratio of Bcl-2/Bax mRNA decreased significantly (P<0.01). In comparison with model group after treatment of acupotomy, Bax mRNA and Caspase-3 mRNA expression decreased significantly (P<0.01, P<0.05) and the ratio of Bcl-2/Bax mRNA increased significantly(P<0.01). In comparison with model group after treatment of electro-acupuncture, there was no significant difference in Bax mRNA and Caspase-3 mRNA expression and the ratio of Bcl-2/Bax mRNA (P>0.05). The results of Real-time PCR tests showed that acupotomy therapy had significant effects in decreasing Bax mRNA and Caspase-3 mRNA expression and upregulating the ratio of Bcl-2/Bax mRNA. The effects of acupotomy group were superior to electro-acupuncture group.5. The results of western blot tests showed that there was no significant difference of Bcl-2 protein between all the groups (P>0.05). In comparison with normal control group after modeling, Bax and Caspase-3 proteins expression increased significantly (P<0.01, P<0.05),and the ratio of Bcl-2/Bax protein decreased significantly (P<0.05). In comparison with model group after treatment of acupotomy, Bax and Caspase-3 proteins expression decreased significantly(P< 0.05) and the ratio of Bcl-2/Bax protein increased significantly(P<0.05). In comparison with model group after treatment of electro-acupuncture, there was no difference in Bax and Caspase-3 protein expression and the ratio of Bcl-2/Bax protein (P> 0.05). The results of western blot tests were consistent to Real-time PCR tests, acupotomy therapy showing great significance in decreasing Bax and Caspase-3 protein expression, and upregulating the ratio of Bcl-2/Bax protein. The effects of acupotomy group were superior to electro-acupuncture group.6. Correlation analysis showed that TUNEL-positive myonuclei count of posterior cervical extensor muscles had a strong positive correlation with cervical X-ray scores after treatment(r=0.832, P<0.01), Bax mRNA expression(r=0.669, P<0.01), Caspase-3 mRNA expression(r=0.644, P<0.01), Bax protein expression(r=0.522, P<0.01), Caspase-3 protein expression (r=0.667, P<0.01). TUNEL-positive myonuclei count of posterior cervical extensor muscles had a strong negative correlation with the ratio of Bcl-2/Bax mRNA(r=-0.613, P< 0.01), ratio of Bcl-2/Bax protein(r=-0.700,P<0.01). There was no correlation between TUNEL-positive myonuclei count of posterior cervical extensor muscles with Bcl-2 mRNA expression(r=-0.145, P>0.05), Bcl-2 protein expression(r=-0.247, P>0.05).Conclusions1. Acupotomy therapy has good effects of regulating myocytes apoptosis in posterior cervical extensor muscles in rabbits with CS, reducing TUNEL-positive myonuclei count.2. Acupotomy therapy can down-regulate genes and proteins expression of Bax and caspase-3, up-regulate the ratio of Bcl-2/Bax mRNA and protein expression in posterior cervical extensor muscles in rabbits with CS. 3. Regulating myocytes apoptosis is the possible mechanism of acupotomy therapy to treat CS and diseases of meridian sinew.
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