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目的通过研究miR-16对神经病理性疼痛模型大鼠脑干中缝核中5羟色胺转运体(SERT)表达的作用,探讨miR-16通过作用于5羟色胺(5HT)再摄取影响神经病理性疼痛的可能机制。方法选用雄性6周龄SD大鼠40只,采用慢性坐骨神经结扎法(chronic constriction injury,CCI)制备大鼠病理性神经痛模型。将大鼠随机分为4组:假手术组(S组),仅分离坐骨神经鞘不结扎;坐骨神经损伤组(CCI组),松弛结扎坐骨神经;坐骨神经损伤+氟西汀组(CCI/F组);对照组(C组),不做任何处理,每组10只。术前1 d及术后第1、3、5、7、14天测定机械缩足痛阈值(MWT);14 d后处死大鼠,取大鼠脑干中缝核组织,通过实时定量PCR法(qRT-PCR)检测miR-16表达情况;western blot检测SERT蛋白表达情况;组织匀浆后,ELISA法检测皮质组织内5羟色胺(5HT)浓度。结果与术前相比,CCI组大鼠第1天MWT即出现下降,与S组相比差异具有统计学意义(t=5.167,P<0.001),CCI/F组与S组相比MWT显著升高,差异具有统计学意义(t=4.664,P<0.001),而S组及C组未见明显变化。qRT-PCR实验显示,与S组相比CCI组大鼠脑干中缝核组织匀浆中内源性miR-16表达显著降低(t=11.840,P<0.001),CCI/F组升高(t=28.640,P<0.001);western blot显示CCI组大鼠脑干中缝核SERT蛋白表达升高,CCI/F组SERT表达降低;ELISA实验显示CCI组皮质中5HT水平较S组显著降低(t=17.920,P<0.001),而CCI/F组5HT水平升高(t=7.232,P=0.002),差异均具有统计学意义。结论 miR-16能够通过抑制脑干中缝核SERT表达,减少5HT再摄取影响病理性神经痛的维持。
Objective To investigate the possible role of miR-16 in 5-HT reuptake on neuropathic pain by studying the role of miR-16 in serotonergic transporter (SERT) expression in the nucleus accumbens of neuropathic pain model rats . Methods Forty male Sprague - Dawley rats of 6 weeks old were used in this study. Chronic constriction injury (CCI) was used to establish the model of pathological neuralgia in rats. The rats were randomly divided into 4 groups: sham operation group (S group), only sciatic nerve sheath was not ligated; sciatic nerve injury group (CCI group), relaxation ligation sciatic nerve; sciatic nerve injury + fluoxetine group (CCI / F group) Control group (C group), without any treatment, each group of 10. Mechanical contractions pain threshold (MWT) was measured on the first day before operation and on the 1st, 3rd, 5th, 7th and 14th days after operation. After 14 days, the rats were sacrificed and the middle part of the brainstem was stained with real-time quantitative PCR qRT-PCR) was used to detect the expression of miR-16. Western blot was used to detect the expression of SERT protein. After homogenate, the concentration of 5HT in cortex was detected by ELISA. Results Compared with preoperative, the MWI of rats in CCI group decreased on the first day, and the difference was statistically significant compared with that of S group (t = 5.167, P <0.001). The MWT of CCI / F group was significantly higher than that of S group The difference was statistically significant (t = 4.664, P <0.001), but there was no significant change in S and C groups. The results of qRT-PCR showed that endogenous miR-16 expression was significantly lower in CCK group than that in S group (t = 11.840, P <0.001), CCI / F group = 28.640, P <0.001). Western blot showed that the expression of SERT protein in the nucleus accumbens of CCI rats increased and the expression of SERT decreased in CCI / F rats. The level of 5HT in cortex of CCI rats was significantly lower than that in rats in CCI rats (t = 17.920, P <0.001), while the 5HT level in CCI / F group was significantly higher (t = 7.232, P = 0.002). The differences were statistically significant. Conclusion miR-16 can inhibit the maintenance of pathological neuralgia by inhibiting the expression of SERT in the nucleus accumbens and reducing the reuptake of 5HT.