蔡磊,方为志,黎清波,等.脉冲电磁场对大鼠椎间盘退变及A2A腺苷受体介导的活性氧/PI3K/Akt信号通路的影响[J].中华物理医学与康复杂志,2022,44(11):966-972
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| 脉冲电磁场对大鼠椎间盘退变及A2A腺苷受体介导的活性氧/PI3K/Akt信号通路的影响 |
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| DOI：10.3760/cma.j.issn.0254-1424.2022.11.002 |
| 中文关键词: 脉冲电磁场 A2A腺苷受体 氧化应激 凋亡 椎间盘退行性病变 |
| 英文关键词: Pulsed electromagnetic fields A2A adenosine receptor Oxidative stress Apoptosis Intervertebral disc degeneration |
| 基金项目:湖北省自然科学基金面上项目（2021CFB522）；武汉市卫生和计划生育委员会一般项目（WX10C20） |
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| 中文摘要: |
| 目的 观察脉冲电磁场（PEMF）对椎间盘退行性病变（IDD）大鼠髓核A2A腺苷受体（A2AR）的调控效应，并探讨其对A2AR介导的活性氧（ROS）/PI3K/Akt信号通路的影响。 方法 采用随机数字表法将50只SD大鼠分为对照组、椎间盘退行性病变组（简称模型组）、A2AR激动剂CGS-21680治疗组（简称激动剂组）、PEMF组和PEMF联合CGS-21680治疗组（简称观察组）。除对照组外，其余各组大鼠均制成IDD动物模型。制模后激动剂组向L5-6椎间盘组织注射100 μl CGS-21680，PEMF组给予PEMF干预，观察组注射CGS-21680后再给予PEMF干预，PEMF干预时间为14 d。培养大鼠原代髓核细胞，将其分为对照组、IL-1β模型组(简称IL-1β组)、激动剂组、PEMF组和观察组，各组细胞干预方法同上。于制模8周后采用HE染色评估各组大鼠椎间盘组织病理形态变化，采用TUNEL染色评估髓核细胞凋亡情况，采用免疫组化/免疫荧光法测定8-OHDG表达，采用ELISA试剂盒等检测超氧化物歧化酶（SOD）、丙二醛（MDA）及ROS含量，采用Western blot技术检测A2AR、PI3K、AKT及p-AKT蛋白水平。 结果 模型组大鼠髓核细胞明显皱缩、坏死，纤维环断裂；观察组纤维环完整，髓核结构基本趋于正常。激动剂组、PEMF组及观察组SOD水平及A2AR、PI3K、p-AKT、AKT蛋白含量均较模型组明显升高，MDA、ROS及8-OHDG表达均显著降低（P＜0.05）；并且观察组ROS水平亦显著低于激动剂组及PEMF组（P＜0.05），p-AKT磷酸化水平则显著高于激动剂组及PEMF组（P＜0.05）。细胞实验结果显示，激动剂组、PEMF组及观察组髓核细胞SOD水平、A2AR、PI3K、p-AKT、AKT蛋白含量均显著高于IL-1β组，MDA、ROS及8-OHDG水平均明显降低（P＜0.05）；并且观察组ROS表达亦显著低于激动剂组和PEMF组（P＜0.05），A2AR蛋白含量及p-AKT磷酸化水平则明显高于激动剂组及PEMF组（P＜0.05）。激动剂组、PEMF组及观察组髓核细胞Bax水平均较IL-1β组显著降低，Bcl-2表达则明显升高（P＜0.05），并且观察组细胞凋亡率亦显著低于激动剂组和PEMF组，Bcl-2表达则显著高于激动剂组及PEMF组（P＜0.05）。 结论 PEMF可通过上调A2AR活性，减少ROS生成，从而发挥抗氧化应激作用；联合A2AR激动剂能进一步激活PI3K/Akt磷酸化，下调促凋亡蛋白Bax水平，上调抗凋亡蛋白Bcl-2表达，从而抑制髓核细胞凋亡，减缓IDD恶性进展。 |
| 英文摘要: |
| Objective To observe any regulatory effect of a pulsed electromagnetic field (PEMF) on A2A adenosine receptors (A2ARs) in the nucleus pulposus of rats with intervertebral disc degeneration (IDD), and to explore any combination with the A2ARs′ agonist-mediating ROS/PI3K/Akt signaling pathway. Methods Fifty Sprague-Dawley rats were randomly divided into a control group, an intervertebral disc degeneration group (the model group), an A2AR agonist CGS-21680 treatment group (the agonist group), a PEMF group and a PEMF combined with CGS-21680 treatment group (the observation group). IDD was modeled in all except the rats in the control group. 100μL of CGS-21680 (100μg/kg) was injected into the L5-6 intervertebral discs of the agonist group, while the PEMF group was given 30 minutes of PEMF intervention daily for 14 days at 1.5mT and 75Hz with a pulse width of 150μs. The observation group was injected with CGS-21680 and then given the same PEMF intervention. Primary nucleus pulposus cells from each group (50ng/mL) were cultured and the expressions of 8-OHDG, SOD, MDA and ROS were detected by immunohistochemistry, immunofluorescence or with an ELISA kit. The A2AR, PI3K, AKT and p-AKT protein levels were detected using western blotting. Results The nucleus pulposus cells and the annulus fibrosus were obviously wrinkled, necrotic and broken in the model group but the annulus fibrosus was intact and the nucleus pulposus was almost normal in the observation group. Compared with the model group, the levels of SOD and A2AR, PI3K, p-AKT and AKT protein were higher in the agonist, PEMF and observation groups, while the expressions of MDA, ROS and 8-OHDG were weaker. The ROS level in the observation group was significantly lower than that in the agonist and PEMF groups, and the phosphorylation level of p-AKT in the observation group was significantly higher than in the agonist and PEMF groups. The average levels of SOD, A2AR, PI3K, p-AKT and AKT protein in the nucleus pulposus cells of the agonist, PEMF and observation groups were significantly higher than the IL-1β group′s average, while the average levels of MDA, ROS and 8-OHDG were significantly lower. The ROS levels in the observation group were significantly lower than in the agonist and PEMF groups, while the A2AR protein content and p-AKT phosphorylation in the observation group were significantly greater. The average Bax levels in the nucleus pulposus cells of the agonist, PEMF and observation groups were significantly lower than that in the IL-1β group, and the expression of Bcl-2 was significantly increased. There was significantly less apoptosis observed in the observation group than in the agonist and PEMF groups, while the expression of Bcl-2 was significantly higher. Conclusions PEMF plays an anti-oxidative stress role by up-regulating A2AR activity and reducing ROS generation. Treatment with PEMF and A2AR agonist could further activate the phosphorylation of PI3K/Akt, down-regulate Bax and up-regulate Bcl-2, thus inhibiting the apoptosis of nucleus pulposus cells and alleviating the malignant progression of IDD. |
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