基于IKKβ/NF-κB通路探究捏脊疗法对颈椎病模型大鼠椎间盘细胞炎症性反应及凋亡的作用

Effect of chiropractic therapy on inflammatory response and apoptosis of intervertebral disc cells in rats with cervical spondylosis based on the IKKβ/NF⁃κB pathway

目的探讨捏脊疗法对颈椎病模型大鼠椎间盘细胞炎症性反应及凋亡的影响及可能的作用机制。方法随机选择8只SPF级SD大鼠作为假手术组(雌雄各半),剩余大鼠利用动静力失衡法诱导椎间盘模型,成模大鼠随机分为模型组、捏脊组及对照组,每组各8只,其中捏脊组每日进行捏脊疗法,对照组大鼠每日灌胃美洛昔康片0.75 mg/kg,假手术组、模型组大鼠不作治疗处理,连续28 d。苏木精-伊红(HE)染色检测椎间盘组织病理学变化;原位末端标记(TUNEL)染色检测椎间盘细胞凋亡情况;酶联免疫吸附(ELISA)法检测血清TNF-α、IL-1β水平;免疫组化技术检测椎间盘组织中肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)蛋白表达水平;实时荧光定量PCR(RT-qPCR)技术检测Caspase-3、B细胞淋巴瘤/白血病-2基因(Bcl2)及Bcl2相关X蛋白(Bax)mRNA相对表达量;蛋白免疫印迹(Western blot)法检测Cleaved Caspase-3、Bax、Bcl2、IκB激酶β(IKKβ)、p-IKKβ、NF-κB抑制蛋白α(IκBα)、p-IκBα、核因子-κBp65(NF-κBp65)及p-p65蛋白表达情况。结果与假手术组比模型组、捏脊组及对照组大鼠椎间盘组织结构均见有不同程度的退化,髓核组织皱缩,与外部纤维环边界不清,椎间盘病理组织学评分、内部髓核(NP)细胞凋亡率、血清TNF-α、IL-1β水平、椎间盘组织中TNF-α、IL-1β蛋白平均光密度值、Caspase-3、Bax mRNA相对表达量、Cleaved Caspase-3、Bax、p-IKKβ、p-p65及p-IκBα蛋白相对表达量升高,椎间盘组织中Bcl2 mRNA及蛋白相对表达量降低(P<0.05);与模型组比较,捏脊组及对照组大鼠椎间盘组织结构退化逐渐减轻,椎间盘病理组织学评分、NP细胞凋亡率、血清TNF-α、IL-1β水平、椎间盘组织中TNF-α、IL-1β蛋白平均光密度值、Caspase-3、Bax mRNA相对表达量、Cleaved Caspase-3、Bax、p-IKKβ、p-p65及p-IκBα蛋白相对表达量降低,椎间盘组织中Bcl2 mRNA及蛋白相对表达量升高(P<0.05);捏脊组上述指标及因子变化程度不及对照组(P<0.05)。结论捏脊疗法可减轻颈椎病大鼠椎间盘细胞炎症性反应及凋亡,改善椎间盘退变(IDD)进展,其作用机制可能与抑制IKKβ/NF-κB通路有关。

Objective To investigate the effect of chiropractic therapy on the inflammatory response and apoptosis of intervertebral disc cells in rats with cervical spondylosis and its possible mechanism.Methods Overall,8 SPF SD rats were randomly selected as the sham operation group(half male and female).The remaining rats were used to establish an intervertebral disc model by the dynamic and static imbalance method.Model rats were randomly divided into model,chiropractic,and control groups with eight rats in each group.The chiropractic group was subjected to chiropractic therapy every day,the control group was administered 0.75 mg/kg meloxicam tablets every day,and sham operation and model groups were untreated for 28 days.HE staining was used to detect histopathological changes of the intervertebral disc.TUNEL staining was used to detect apoptosis of intervertebral disc cells.Immunohistochemistry was used to detect tumor necrosis factor(TNF⁃α)and interleukin⁃1β(IL⁃1β)protein expression in intervertebral disc tissue.Serum TNF⁃αand IL⁃1βlevels were measured by ELISA.RT⁃qPCR was used to measure relative mRNA expression of Caspase⁃3,B⁃cell lymphoma/leukemia⁃2 gene(Bcl2)and Bcl2⁃related X protein(Bax).Protein expression of Cleaved Caspase⁃3,Bax,Bcl2 and IκB kinaseβ(IKKβ),p⁃IKKβ,NF⁃κB inhibitor proteinα(IκBα),p⁃IκBα,nuclear factor⁃κB p65(NF⁃κB p65)and p⁃p65 was detected by Western blot.Results Compared with the sham operation group,the model,chiropractic,and control groups showed that the tissue structure of intervertebral disc was in various degrees of degeneration,the nucleus pulposus tissue shrinked,and the boundary with the external fibrous ring was unclear,meanwhile,intervertebral disc histopathological score,nucleus pulposus(NP)cell apoptosis rate,serum TNF⁃αand IL⁃1β,TNF⁃αand IL⁃1βprotein expression,Caspase⁃3 and Bax mRNA expression,and p⁃IKKβ,p⁃p65 and p⁃IκBαprotein expression in the intervertebral disc tissue all increased,while the expression of Bcl2 mRNA and protein decreaed(P<0.05).Compared with the model group,the chiropractic,and control groups showed that degeneration of the intervertebral disc tissue structure was reduced gradually,and the intervertebral disc histopathological score,NP cell apoptosis rate serum TNF⁃αand IL⁃1β,TNF⁃αand IL⁃1βprotein expression,caspase⁃3 and Bax mRNA expression,and p⁃IKKβ,p⁃p65 and p⁃IκBαprotein expression in the intervertebral disc tissue were decreased,while Bcl2 mRNA and protein expression were increased in intervertebral disc tissue(P<0.05).Changes in the above indexes and factors in the chiropractic group were lower than those in the control group(P<0.05).Conclusions Chiropractic therapy reduces the inflammatory reaction and apoptosis of intervertebral disc cells in rats with cervical spondylosis and improves the progression of intervertebral disc degeneration.The mechanism may be related to inhibition of the IKKβ/NF⁃κB pathway.