侧脑室注射miRNA-22对癫痫大鼠海马P2X7受体及炎性因子表达的影响

Effect of intracerebroventricular injection of miRNA-22 on expression of P2X7 receptor and inflammatory factors in hippocampus of rats with epilepsy

目的探讨调控基因microRNA-22(miRNA-22)对癫痫大鼠海马组织中P2X7受体及炎性因子表达的影响。方法健康SD雄性大鼠,采用腹腔注射氯化锂-匹罗卡品诱导癫痫大鼠模型。选取造模成功3 d后的大鼠45只,按随机数字表法分成3组,分别为癫痫组、miRNA-22 agomir组和miRNA-22 agomir control组,另选15只正常大鼠作为对照,应用Western-blot检测各组P2X7受体的表达情况,qRT-PCR检测各组miRNA-22、P2X7 mRNA、NF-κB mRNA和IL-1β mRNA的表达,尼氏染色观察尼氏小体损伤情况。结果Western-blot实验结果发现,与正常组(0.91±0.10)相比,癫痫组大鼠致痫灶内P2X7受体(1.17±0.05)的表达水平是增高的,差异具有统计学意义(t=-4.11,P=0.02);miRNA-22 agomir组,P2X7的表达(0.66±0.06)较miRNA-22 agomir control组(0.94±0.14)是降低的,差异具有统计学意义(t=-3.10,P=0.04)。qRT-PCR实验结果发现,与正常组相比,癫痫组大鼠致痫灶内P2X7 mRNA(9.08±0.94)、NF-κB mRNA(20.10±2.15)和IL-1β mRNA(50.64±5.42)的表达水平增高,差异具有统计学意义(t=-14.96,P<0.05;t=-15.38,P<0.05;t=-15.87,P<0.05)。miRNA-22 agomir组P2X7 mRNA(1.31±0.64)、NF-κB mRNA(2.28±1.10)和IL-1β mRNA(2.12±1.20)的表达较癫痫组[(9.08±0.94),(20.10±2.15),(50.64±5.42)]、miRNA-22 agomir control组[(7.03±1.90),(18.72±1.76),(47.39±6.16)]明显降低,差异具有统计学意义(F=29.77,P<0.05;F=98.99,P<0.05;F=96.29,P<0.05)。尼氏染色实验结果发现,在癫痫大鼠海马CA1、CA3区,镜下能够观察到大量形态异常、解体的尼氏小体,表现为体积变小,形态呈不规则或三角形,排列紊乱,空泡样变性,与正常组大鼠相比,差异有统计学意义(P<0.05);侧脑室注射miRNA-22 agomir后检测到尼氏小体解体情况有所改善,尼氏小体计数增多。结论侧脑室注射miRNA-22 agomir能够下调癫痫大鼠海马组织中P2X7受体及相关炎症因子的表达,从而抑制癫痫发作。

Objective To investigate the effect of microRNA-22 (miRNA-22) on the expression of P2X7 receptor and inflammatory factors in hippocampus of rats with epilepsy. Methods Healthy SD male rats were intraperitoneal injected with lithium chloride and pilocarpine to induce epilepsy. Three days later, 45 epileptic rats were randomly divided into three groups: epilepsy group(EP group), miRNA-22 agomir group (EF+ agomir group)and miRNA-22 agomir control group(EF+ agomir control group). Another 15 healthy rats were selected as control group(N group). The expression of P2X7 protein was detected by Western blot and the levels of miRNA-22, P2X7 mRNA, NF-κB mRNA , IL-1β mRNA were detected by qRT-PCR. Nissl staining was used to observe the damage of Nissl bodies. Results Western blot result showed that compared with the N group(0.91±0.10), the level of P2X7 protein in EP group (1.17±0.052) increased, and the difference was statistically significant (t=-4.11, P=0.02). Compared with the EP+ agomir control group(0.94±0.14), the expression of P2X7 protein in EP+ agomir group (0.66±0.06) decreased and the difference was significant (t=-3.10, P=0.04). And the qRT-PCR results showed that compared with N group, the levels of P2X7mRNA (9.08±0.94), NF-κB mRNA (20.10±2.15) and IL-1β mRNA (50.64±5.42) in EP group increased(t=-14.96, P<0.05;t=-15.38, P<0.05;t=-15.87, P<0.05). The expression of P2X7mRNA (1.31±0.64), NF-κB mRNA (2.28±1.10) and IL-1β mRNA (2.12±1.20) in EF+ agomir group decreased compared with EP group((9.08±0.94),(20.10±2.15),(50.64±5.42)) and EF+ agomir control group((7.03 ±1.90),(18.72±1.76),(47.39±6.16)), and the differences were statistically significant(F=29.77, P<0.01;F=98.99, P<0.05;F=96.29, P<0.01). Nissl staining results showed that a large number of morphologically abnormal and disintegrated Nissl bodies could be observed in the hippocampal CA1 and CA3 regions of EP group, which showed a smaller size, irregular morphology, chromatin pyknosis, boundary blur between nucleus and cytoplasm. Compared with the normal group, the difference was significant (P<0.05). While in miRNA-22 agomir group, the disintegration of Nissl bodies was improved and the number of Nissl bodies increased. Conclusion Intraventricular injection of miRNA-22 agomir can down-regulate the expression of P2X7 receptor and related inflammatory factors in hippocampus of epileptic rats, thus inhibiting seizures.