摘要
目的观察脉冲电磁场(PEMF)联合A_(2A)腺苷受体激动剂CGS-21680对大鼠椎间盘退变髓核细胞凋亡及炎性反应的影响。方法采用随机数字表法将48只SD大鼠分为假手术组、模型组、A_(2A)腺苷受体激动剂CGS-21680治疗组(简称激动剂组)和PEMF联合CGS-21680治疗组(简称观察组)。除假手术组外,其余各组均制成椎间盘退行性病变(IDD)大鼠模型。制模后激动剂组向L5-6椎间盘注射100μl CGS-21680(100μg/kg),观察组注射CGS-21680后再给予PEMF干预,磁场强度1.5 mT,磁场频率75 Hz,脉宽150μs,暴磁30 min/次/d,连续干预14 d,假手术组及模型组同期注射等量生理盐水。于实验进行8周后采用HE染色观察椎间盘组织病理形态变化,采用免疫组化法检测Ⅱ型胶原(Col-Ⅱ)蛋白表达,采用ELISA及RT-PCR法检测炎性因子白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)含量及mRNA表达,采用Western blot及RT-PCR法检测A_(2A)R、NLRP3、Caspses-3蛋白含量及mRNA水平。结果模型组髓核及纤维环退变明显,观察组髓核细胞皱缩、坏死及纤维环断裂情况显著缓解。干预后观察组椎间盘髓核ColⅡ阳性表达、A_(2A)R蛋白含量及mRNA相对表达量均较模型组、激动剂组明显增加(P<0.05);而促炎因子IL-6、TNF-α水平及mRNA表达量均较模型组、激动剂组显著降低(P<0.05);另外观察组大鼠椎间盘组织NLRP3、Caspase-3蛋白含量及mRNA相对表达量亦较模型组、激动剂组明显降低(P<0.05)。结论PEMF联合A_(2A)腺苷受体激动剂CGS-21680能协同上调IDD模型大鼠A_(2A)R活性,下调促炎因子IL-6、TNF-α及NLRP3、Caspase-3表达,抑制髓核细胞凋亡,减轻炎性反应,有助于椎间盘退变病情缓解。
Objective To explore the effect of combining pulsed electromagnetic field(PEMF)stimulation with A_(2A) adenosine receptor agonist CGS-21680 on apoptosis and inflammation of nucleus pulposus cells in cases of intervertebral disc degeneration.Methods Forty-eight Sprague-Dawley rats were randomly divided into a sham operation group(Sham group),an intervertebral disc degenerative disease group(Model group),an A_(2A) adenosine receptor agonist CGS-21680 group(Agonist group),and a group in which PEMF was combined with CGS-21680(Observation group).The rat model of intervertebral disc degeneration(IDD)was established in all other groups than the sham operation group.The rats in the Agonist group were injected with 100μL of CGS-21680(100μg/kg)at the L5-6 intervertebral disc.The Observation group was injected with CGS-21680 similarly but then received 14 conse-cutive days of PEMF stimulation(30min/time).The Sham and Model groups were injected with the same amount of normal saline solution.Eight weeks later,HE staining was used to evaluate the pathological changes in the intervertebral disc tissues.The expression of type II collagen was determined by immunohistochemistry.The content and mRNA expression of inflammatory factors interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were determined using enzyme-linked immunosorbent assays and reverse transcription-polymerase chain reactions(RT-PCRs).The protein and mRNA levels of A_(2A),NLRP3 and caspase-3 were determined by western blotting and RT-PCR.Results The degeneration in the nucleus pulposus and annulus fibrosus in the Model group was significant,while significantly less shrinkage,necrosis and fibrous annulus rupture was observed in the Observation group.Compared with Model and Agonist groups,the positive expression of ColⅡin the nucleus pulposus,A_(2A)R protein levels and relative expression of its mRNA had all increased significantly in the Observation group,while the average levels and mRNA expression of pro-inflammatory cytokines IL-6 and TNF-αhad decreased significantly.The average protein level and mRNA expression of NLRP3 and caspase-3 in the intervertebral disc tissues of the Observation group were significantly lower than in the Model and Agonist groups.Conclusions Combining pulsed electromagnetic field stimulation with A_(2A) adenosine receptor agonist CGS-21680 can inhibit the apoptosis of nucleus pulposus cells,alleviate disease response and delay IDD by up-regulating the activity of A_(2A) receptors and down-regulating the expressions of pro-inflammatory factors IL-6,TNF-α,NLRP3 and caspase-3 in nucleus pulposus cells.
作者
刘伟军
焦红博
王威
黎清波
蔡磊
王正坤
Liu Weijun;Jiao Hongbo;Wang Wei;Li Qingbo;Cai Lei;Wang Zhengkun
出处
《中华物理医学与康复杂志》
CAS
CSCD
北大核心
2021年第8期673-679,共7页
Chinese Journal of Physical Medicine and Rehabilitation
基金
湖北省自然科学基金面上项目(2018CFB720)。
关键词
脉冲电磁场
A_(2A)腺苷受体
椎间盘退行性病变
凋亡
炎性反应
Electromagnetic stimulation
A_(2A)adenosine receptor
Intervertebral disc degeneration
Apoptosis
Inflammation
