腺苷受体2A调控神经肽Y抑制髓核细胞凋亡及基质降解的机制研究

Adenosine A2A receptor regulation of neuropeptide Y to inhibit nucleus pulposus cell apoptosis and matrix degradation

目的探索腺苷受体2A(A2AR)调控神经肽Y(NPY)的机制及其在椎间盘退行性病变中对髓核细胞凋亡和细胞外基质的影响。方法采用随机数字表分组法将30只雄性SD大鼠分为5组,每组6只。S组为对照组,即穿刺大鼠L5~6椎间盘后注射生理盐水;M组为模型组,即通过椎间盘穿刺联合椎间盘内注射肿瘤坏死因子α构建大鼠椎间盘退变模型;A2AR-小干扰RNA(siRNA)组、NC-siRNA组和CGS-21680组则为构建椎间盘退变模型基础上分别向椎间盘内注射A2AR的siRNA、阴性对照(NC)siRNA和A2AR激动剂CGS-21680。采用蛋白质印迹法和实时荧光定量聚合酶链反应检测A2AR、NPY、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、Ⅱ型胶原(Col-Ⅱ)、蛋白激酶A(PKA)和环磷酸腺苷反应元件结合蛋白(CREB)等蛋白在椎间盘中的表达。组间比较采用单因素方差分析。结果在M组中,A2AR(1.72±0.50比0.95±0.18,F=11.49,P>0.05)、NPY(0.40±0.03比0.20±0.04,F=22.08,P<0.05)和bax(0.62±0.52比0.40±0.04,F=30.97,P<0.01)蛋白表达水平高于S组,而Col-Ⅱ(0.39±0.03比0.75±0.08,F=16.24,P<0.01)低于S组;在CGS-21680组中,A2AR(2.70±0.45比1.72±0.50,F=11.49,P<0.05),NPY(0.55±0.05比0.40±0.03,F=22.08,P<0.05)和Col-Ⅱ(0.53±0.11比0.39±0.03,F=16.24,P>0.05)表达高于M组,而bax表达低于M组(0.48±0.08比0.62±0.52,F=30.97,P>0.05);在A2AR-siRNA组中,各分子表达变化与CGS-21680组相比与M组相反:A2AR(0.95±0.29比1.72±0.50,F=11.49,P>0.05),NPY表达(0.20±0.06比0.40±0.03,F=22.08,P<0.01),bax(0.72±0.09比0.85±0.04,F=28.01,P>0.05)和Col-Ⅱ(0.42±0.14比0.59±0.19,F=21.62,P>0.05)。结论A2AR可能通过PKA/CREB信号通路上调NPY及其受体起到减少髓核细胞凋亡和保护细胞外基质的作用。

Objective To explore the mechanism of adenosine receptor 2A(A2AR)regulation on neuropeptide Y(NPY)and its impact on nucleus pulposus cells(NPCs)apoptosis and extracellular matrix(ECM)in intervertebral disc degeneration(IVDD).Methods The total 30 male SD rats were divided into 5 groups by a random number table grouping method,with 6 rats in each group.Group S was control group,in which normal saline was injected after puncturing the L5-6 disc.Group M was model group,in which the rat intervertebral disc degeneration(IDD)model was established through disc puncture combined with intradiscal injection of tumor necrosis factor-alpha(TNF-α).Group A2AR-small interfering RNA(siRNA),group negative control(NC)-siRNA,and group CGS-21680 were subsequently established,in which A2AR-siRNA,NC-siRNA,and A2AR agonist CGS-21680 were respectively injected into the disc based on the established IDD model.Western blotting and real-time quantitative polymerase chain reaction were used to detect the expression of A2AR,NPY,bcl-2-associated X protein(bax),collagen typeⅡ(Col-Ⅱ),protein kinase A(PKA)and cAMP response element-binding protein(CREB)in IVD.One-way analysis of variance was used for inter-group comparisons.Results In group M,A2AR(1.72±0.50 vs.0.95±0.18,F=11.49,P>0.05),NPY(0.40±0.03 vs.0.20±0.04,F=22.08,P<0.05),and bax(0.62±0.52 vs.0.40±0.04,F=30.97,P<0.01)exhibited higher protein expression levels than in group S,while Col-Ⅱ(0.39±0.03 vs.0.75±0.08,F=16.24,P<0.01)displayed lower expression.In group CGS-21680,A2AR(2.70±0.45 vs.1.72±0.50,F=11.49,P<0.05),NPY(0.55±0.05 vs.0.40±0.03,F=22.08,P<0.05)showed increased expression compared to group M,whereas bax(0.48±0.08 vs.0.62±0.52,F=30.97,P>0.05)demonstrated reduced expression.In group A2AR-siRNA,the expression trends for each molecule were opposite to those in group CGS-21680 compared to group M:A2AR(0.95±0.29 vs.1.72±0.50,F=11.49,P>0.05),NPY(0.20±0.06 vs.0.40±0.03,F=22.08,P<0.01),bax(0.72±0.09 vs.0.85±0.04,F=28.01,P>0.05),and Col-Ⅱ(0.42±0.14 vs.0.59±0.19,F=21.62,P>0.05).Conclusion A2AR may upregulate NPY/NPY2R through the PKA/CREB signaling pathway to alleviate NPCs apoptosis and protect ECM.