Liu Pei-Wen, Yue Ming-Xia, Zhou Rui, Niu Juan, Huang Du-Juan, Xu Tao, Luo Pei, Liu Xiao-Hong, Zeng Jun-Wei
Department of Physiology, Zunyi Medical College, Guizhou, China.
J Pain Res. 2017 Jul 26;10:1755-1767. doi: 10.2147/JPR.S137131. eCollection 2017.
P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y and P2Y receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y and P2Y receptors activation is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPβs (ADP analog) on the expression and the release of IL-1β, IL-6, and TNF-α.
In this study, we observed the effect of P2Y receptor agonist ADPβs on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPβs induced the increased expression of Iba-1, IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPβs-evoked increase in Iba-1, IL-1β, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y receptor antagonist MRS2395 or P2Y receptor antagonist MRS2211, respectively. Similarly, ADPβs-evoked release of IL-1β, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPβs-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y and P2Y receptor-evoked increased gene expression of IL-1β, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y and P2Y receptor-evoked release of IL-1β, IL-6 and TNF-α, were also inhibited by Y-27632, SB203580 and PDTC, respectively.
These observations suggest that P2Y and P2Y receptor-evoked gene expression and release of IL-1β, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.
P2受体因其对小胶质细胞中神经兴奋性物质的反应，而与神经递质和促炎细胞因子的释放有关。背角P2Y和P2Y受体参与了由周围神经损伤诱导的疼痛行为的发展。然而，尚不清楚P2Y和P2Y受体激活是否与培养的脊髓背角小胶质细胞中白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）的表达和释放相关。因此，我们研究了ADPβS（ADP类似物）对IL-1β、IL-6和TNF-α表达和释放的影响。
在本研究中，我们使用实时荧光定量聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）观察P2Y受体激动剂ADPβS对IL-1β、IL-6和TNF-α表达和释放的影响。ADPβS在信使核糖核酸（mRNA）水平诱导Iba-1、IL-1β、IL-6和TNF-α表达增加。ADPβS引起的Iba-1、IL-1β、IL-6和TNF-α mRNA表达增加分别仅被P2Y受体拮抗剂MRS2395或P2Y受体拮抗剂MRS2211部分抑制。同样，ADPβS引起的IL-1β、IL-6和TNF-α释放仅被MRS2395或MRS2211部分抑制。此外，在联合使用MRS2395加MRS2179后，ADPβS引起的Iba-1、IL-1β、IL-6和TNF-α mRNA表达增加以及IL-1β、IL-6和TNF-α释放几乎全部被阻断。进一步的证据表明，P2Y和P2Y受体引起的IL-1β、IL-6和TNF-α基因表达增加分别被Y-27632（ROCK抑制剂）、SB203580（P38丝裂原活化蛋白激酶抑制剂）和PDTC（NF-κB抑制剂）抑制。随后，P2Y和P2Y受体引起的IL-1β、IL-6和TNF-α释放也分别被Y-27632、SB203580和PDTC抑制。
这些观察结果表明，P2Y和P2Y受体引起的IL-1β、IL-6和TNF-α基因表达和释放与ROCK/P38丝裂原活化蛋白激酶/NF-κB信号通路有关。
