三维平衡正脊手法联合针刺干预椎间盘退变模型大鼠作用机制研究

Study on Mechanism of Three-Dimensional Balanced Spine Manipulation Combined with Acupuncture Intervention in Model Rats with Intervertebral Disc Degeneration

目的:探讨三维平衡正脊手法联合针刺对椎间盘退变(IVDD)模型大鼠的作用机制。方法:将36只SD大鼠按随机数字表法分为假手术组、模型组、针刺组、联合治疗组,每组9只。模型组、针刺组、联合治疗组以尿激酶型纤溶酶原激活物(uPA)注射法建立IVDD模型,假手术组按造模流程切开大鼠软组织,但不注射uPA。造模成功后,模型组、假手术组不进行任何干预,针刺组采用针刺治疗,联合治疗组采用三维平衡正脊手法联合针刺,持续干预6周。于实验结束后观察大鼠目标椎间盘组织形态变化,并检测大鼠椎间盘组织白细胞介素-1β(IL-1β)蛋白及蛋白聚糖、Ⅱ型胶原(Col2A1)及基质金属蛋白酶-3(MMP-3)mRNA的表达情况。结果:各组大鼠椎间盘组织形态有一定差异,苏木精-伊红(HE)染色结果显示假手术组大鼠椎间盘髓核及纤维环结构最为完整,模型组大鼠椎间盘基本不可见髓核组织,纤维环可见大量放射状破裂样痕迹,针刺组及联合治疗组大鼠椎间盘纤维环可见少量破裂样痕迹及保留的髓核组织,且联合治疗组椎间盘结构相对完整。与假手术组比较,模型组大鼠IL-1β蛋白表达水平显著升高(P<0.05),蛋白聚糖、Col2A1 mRNA表达水平显著降低(P<0.05),MMP-3 mRNA表达水平显著上升(P<0.05)。与模型组比较,针刺组和联合治疗组IL-1β蛋白表达水平下降(P<0.05),蛋白聚糖、Col2A1 mRNA表达水平上升(P<0.05);针刺组MMP-3 mRNA表达水平下降,但差异无统计学意义(P>0.05);联合治疗组MMP-3 mRNA表达水平显著下降(P<0.05)。与针刺组比较,联合治疗组IL-1β蛋白表达水平显著下降(P<0.05),蛋白聚糖、Col2A1 mRNA表达水平显著上升(P<0.05),MMP-3 mRNA表达水平显著下降(P<0.05)。结论:①uPA注射可以诱导大鼠IVDD。②IVDD模型大鼠椎间盘IL-1β、MMP-3、Col2A1、蛋白聚糖表达水平发生改变,IL-1β、MMP-3、Col2A1、蛋白聚糖可参与椎间盘退变。③三维平衡正脊手法联合针刺可促进IVDD模型大鼠椎间盘形态学恢复,并可通过调节IL-1β、MMP-3、Col2A1、蛋白聚糖的表达干预椎间盘退变。④三维平衡正脊手法联合针刺对IVDD的干预作用优于单纯针刺干预。

Objective:To investigate the mechanism of three-dimensional balanced spine manipulation combined with acupuncture in model rats with intervertebral disc degeneration(IVDD).Methods:Thirty-six SD rats were divided into sham operation group,model group,acupuncture group and combined treatment group according to the random number table method,with 9 rats in each group.The IVDD model was established by urokinase-type fibrinogen activator(uPA)injection in the model group,acupuncture group and combined treatment group,and the soft tissues of rats in the sham operation group were cut according to the modeling procedure,but not injected with uPA.After the successful modeling,the model group and the sham operation group did not receive any intervention,the acupuncture group was treated with needling,and the combined treatment group was treated with three-dimensional balanced spine manipulation combined with needling,the intervention lasted for 6 weeks.At the end of the experiment,we observed the morphological changes of the target intervertebral discs in rats and detected the expression levels of interleukin-1β(IL-1β)protein and proteoglycan,typeⅡcollagen(Col2A1)and matrix metalloproteinase-3(MMP-3)mRNA.Results:The morphology of the intervertebral discs of rats in each group showed some differences.Hematoxylineosin staining(HE staining)showed that the nucleus pulposus and annulus fibrosus of the intervertebral discs in the sham operation group were the most intact,while the model group had no nucleus pulposus,and the annulus fibrosus of the model group had a lot of radial rupture traces,and the annulus fibrosus of the acupuncture group and the combined treatment group had a few rupture traces and preserved nucleus pulposus,while the structure of the intervertebral disc in the combined treatment group was more complete.Compared with the sham operation group,the protein expression level of IL-1βin the model group was increased significantly(P<0.05),proteoglycan and Col2A1 mRNA expression levels were decreased significantly(P<0.05),and the expression level of MMP-3 mRNA was significantly raised(P<0.05).Compared with the model group,IL-1βprotein expression level was lowered in the acupuncture group and combined treatment group(P<0.05),proteoglycan and Col2A1 mRNA expression levels were raised(P<0.05).MMP-3 mRNA expression level was reduced in the acupuncture group,there was no statistical significance(P>0.05);MMP-3 mRNA expression level was decreased significantly in the combined treatment group(P<0.05).Compared with the acupuncture group,IL-1βprotein expression level was reduced significantly in the combined treatment group(P<0.05),proteoglycan and Col2A1 mRNA expression levels were raised significantly(P<0.05),and MMP-3 mRNA expression level was decreased significantly(P<0.05).Conclusions:①uPA injection can induce IVDD in rats.②The expression levels of IL-1β,MMP-3,Col2A1 and proteoglycan were altered in the intervertebral discs of rats in the IVDD model,and IL-1β,MMP-3,Col2A1 and proteoglycan could be involved in intervertebral disc degeneration.③Three-dimensional balanced spine manipulation combined with acupuncture can promote the morphological recovery of intervertebral discs in IVDD model rats,and can intervene in intervertebral disc degeneration by regulating the expression levels of IL-1β,MMP-3,Col2A1 and proteoglycan.④The intervention effect of three-dimensional balanced spine manipulation combined with acupuncture on intervertebral disc degeneration was superior to that of acupuncture intervention alone.