顆粒性白血球群落刺激因子於慢性壓迫性神經損傷大鼠之止痛機轉
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2022
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位於周邊神經，背根神經節 (dorsal root ganglia，DRGs)及脊髓背角細胞(spinal dorsal horn，SDH)的各種不同發炎性介質，包含μ類鴉片受體 (mu-opioid receptor，MOR)、促炎性/抗炎性細胞素(pro-inflammatory/anti-inflammatory cytokine)、趨化素(chemokine)、小分子核糖核酸(microribonucleic acid，microRNA)和磷酸化-p38 (phospho-p38，p-p38) 在神經性疼痛的生成均伴有重要的角色。此外，細胞自噬(autophagy)及細胞凋亡(apoptosis)也調節了神經性疼痛的形成。顆粒性白血球群落刺激因子 (granulocyte colony-stimulating factor，G-CSF) 是一種生長因子，可刺激周邊血液中顆粒性白血球的形成，對神經性疼痛有鎮痛的作用。它是經由聚集含鴉片類物質的白血球到受損神經處，並抑制DRGs上的pro-inflammatory cytokine來達成止痛效果。此外，G-CSF也以多種方式對microRNA的表現、autophagy及apoptosis的活性產生影響。然而，G-CSF詳細的鎮痛機轉，以及pro-inflammatory cytokine、chemokine、microRNA、autophagy和apoptosis在慢性神經疼痛形成中的角色則尚未完全明瞭。因此，我們藉由動物疼痛行為測試，西方墨點法，酵素免疫分析法和免疫組織化學方法在神經損傷後的不同時間點（分別為神經損傷後1、3 和 7 天）分析假手術，接受與非接受G-CSF治療的慢性壓迫性神經損傷大鼠之受損周邊神經及DRGs上MOR、pro-inflammatory cytokine、chemokine、microRNA、autophagy和apoptosis蛋白質，及SDH上p-p38和pro-inflammatory cytokine的表現。結果顯示，在神經損傷後給予單次全身性的G-CSF治療後，可在神經受傷的初期促進受損周邊神經及DRGs上的MOR、microRNA-122、和細胞自噬蛋白質(autophagy protein: microtubule-associated protein light chain 3-II ，LC3II)的表達。然後這一系列的變化不但抑制了DRGs上的pro-inflammatory cytokine及chemokine (monocyte chemoattractant protein-1，MCP-1)的表現，並且在神經受傷的後期抑制了DRGs上的apoptosis蛋白質的表現，以及抑制SDH上的p-p38、pro-inflammatory cytokine (interleukin-6，IL-6) 的活性；唯增強了SDH上anti-inflammatory (interleukin-4，IL-4)的表現，藉此減輕神經性疼痛。因此，G-CSF 可以作為調節受損周邊神經、DRGs、SDH上pro-inflammatory cytokine、chemokine、microRNA、autophagy和apoptosis蛋白質表達的藥物，並進一步成為具有治療神經性疼痛潛力的藥物。然而，autophagy的神經性疼痛調節作用具有時間依賴性，必須在pro-inflammatory cytokine達到誘發神經性疼痛的閾值前的神經受傷初期階段，增加autophagy活性才可以有效抑制pro-inflammatory cytokine和apoptosis蛋白質的表達，藉此緩解神經性疼痛的進一步的發展。
The different inflammatory mediators including the mu-opioid receptor (MOR), pro-inflammatory/anti-inflammatory cytokines, chemokines, microribonucleic acid (microRNA), and phospho-p38 (p-p38) located on the peripheral nerves, the dorsal root ganglia (DRGs), and the spinal dorsal horn (SDH) have critical roles in neuropathic pain development. In addition to the previous agents, both autophagic and apoptotic activities can also modulate neuropathic pain formation. Granulocyte colony-stimulating factor (G-CSF) is a growth factor that can promote granulocyte production and has analgesic effects on neuropathic pain. The analgesic effects of G-CSF occur through recruiting leukocytes that secrete opioids to the injured nerve and suppressing pro-inflammatory cytokines in the DRGs. In addition, G-CSF can also modulate microRNA expressions, as well as autophagic and apoptotic activities in many ways. However, the detailed underlying analgesic mechanisms of G-CSF and the roles of pro-inflammatory cytokines, chemokines, microRNAs, autophagy, and apoptosis in chronic neuropathic pain formation are not fully understood. Therefore, mechanical allodynia, various levels of MORs, pro-inflammatory cytokines, chemokines, microRNAs, and apoptotic and autophagic proteins in the damaged sciatic nerves, and DRGs, p-p38, and different cytokine levels in the SDH were studied in the sham control. Rats with chronic constriction injuries received vehicle or G-CSF treatments at different times after nerve damage (1, 3, and 7 days) using Western blot analysis, enzyme-linked immunosorbent assays, and immunohistochemistry methods. The results demonstrated that a single intravenous dose of G-CSF immediately after nerve damage could increase MOR, microRNA-122, and autophagic protein (microtubule-associated protein light chain 3-II, LC3-II) expressions in the injured nerves and DRGs at an early time point after nerve damage. After that, those alterations suppressed pro-inflammatory cytokine and chemokine (monocyte chemoattractant protein-1, MCP-1) expressions and then suppressed the expressions of apoptotic proteins in the DRGs at a late time point after nerve damage. This further decreased p-p38 and pro-inflammatory cytokine (interleukin-6, IL-6) activities but enhanced anti-inflammatory cytokine (interleukin-4, IL-4) expressions in the SDH, and attenuated neuropathic pain. Thus, medications such as G-CSF could modulate the expressions of different pro-inflammatory cytokines, chemokines, microRNAs, and autophagic and apoptotic proteins in the injured nerve, DRGs, and SDH, which could potentially suppress neuropathic pain formation. However, this autophagy-mediated modulation of pain formation is time-dependent. Increased autophagy before pro-inflammatory cytokines reach the threshold level to promote neuropathic pain formation could effectively suppress pro-inflammatory cytokine expressions and apoptotic activities and alleviate the further development of neuropathic pain.
The different inflammatory mediators including the mu-opioid receptor (MOR), pro-inflammatory/anti-inflammatory cytokines, chemokines, microribonucleic acid (microRNA), and phospho-p38 (p-p38) located on the peripheral nerves, the dorsal root ganglia (DRGs), and the spinal dorsal horn (SDH) have critical roles in neuropathic pain development. In addition to the previous agents, both autophagic and apoptotic activities can also modulate neuropathic pain formation. Granulocyte colony-stimulating factor (G-CSF) is a growth factor that can promote granulocyte production and has analgesic effects on neuropathic pain. The analgesic effects of G-CSF occur through recruiting leukocytes that secrete opioids to the injured nerve and suppressing pro-inflammatory cytokines in the DRGs. In addition, G-CSF can also modulate microRNA expressions, as well as autophagic and apoptotic activities in many ways. However, the detailed underlying analgesic mechanisms of G-CSF and the roles of pro-inflammatory cytokines, chemokines, microRNAs, autophagy, and apoptosis in chronic neuropathic pain formation are not fully understood. Therefore, mechanical allodynia, various levels of MORs, pro-inflammatory cytokines, chemokines, microRNAs, and apoptotic and autophagic proteins in the damaged sciatic nerves, and DRGs, p-p38, and different cytokine levels in the SDH were studied in the sham control. Rats with chronic constriction injuries received vehicle or G-CSF treatments at different times after nerve damage (1, 3, and 7 days) using Western blot analysis, enzyme-linked immunosorbent assays, and immunohistochemistry methods. The results demonstrated that a single intravenous dose of G-CSF immediately after nerve damage could increase MOR, microRNA-122, and autophagic protein (microtubule-associated protein light chain 3-II, LC3-II) expressions in the injured nerves and DRGs at an early time point after nerve damage. After that, those alterations suppressed pro-inflammatory cytokine and chemokine (monocyte chemoattractant protein-1, MCP-1) expressions and then suppressed the expressions of apoptotic proteins in the DRGs at a late time point after nerve damage. This further decreased p-p38 and pro-inflammatory cytokine (interleukin-6, IL-6) activities but enhanced anti-inflammatory cytokine (interleukin-4, IL-4) expressions in the SDH, and attenuated neuropathic pain. Thus, medications such as G-CSF could modulate the expressions of different pro-inflammatory cytokines, chemokines, microRNAs, and autophagic and apoptotic proteins in the injured nerve, DRGs, and SDH, which could potentially suppress neuropathic pain formation. However, this autophagy-mediated modulation of pain formation is time-dependent. Increased autophagy before pro-inflammatory cytokines reach the threshold level to promote neuropathic pain formation could effectively suppress pro-inflammatory cytokine expressions and apoptotic activities and alleviate the further development of neuropathic pain.
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神經性疼痛, 慢性壓迫性損傷, 顆粒性白血球群落刺激因子, μ型類鴉片受體, 發炎性細胞素, 趨化素, 小分子核糖核酸, 細胞自噬, 細胞凋亡, 磷酸化-p38, neuropathic pain, chronic constriction injury, granulocyte colony-stimulating factor (G-CSF), mu-opioid receptor (MOR), pro-inflammatory cytokine, chemokine, microribonucleic acid (microRNA), autophagy, apoptosis, phospho-p38 (p-p38)