BACKGROUND Neuroinflammation plays a vital role in the development and maintenance of neuropathic pain. Recent evidence has proved that bone marrow mesenchymal stem cells (BMSCs) can inhibit neuropathic pain and possess potent immunomodulatory and immunosuppressive properties via secreting a variety of bioactive molecules, such as TNF-α-stimulated gene 6 protein (TSG-6). However, it is unknown whether BMSCs exert their analgesic effect against neuropathic pain by secreting TSG-6. Therefore, the present study aimed to evaluate the analgesic effects of TSG-6 released from BMSCs on neuropathic pain induced by chronic constriction injury (CCI) in rats and explored the possible underlying mechanisms in vitro and in vivo. METHODS BMSCs were isolated from rat bone marrow and characterized by flow cytometry and functional differentiation. One day after CCI surgery, about 5 × 106 BMSCs were intrathecally injected into spinal cerebrospinal fluid. Behavioral tests, including mechanical allodynia, thermal hyperalgesia, and motor function, were carried out at 1, 3, 5, 7, 14 days after CCI surgery. Spinal cords were processed for immunohistochemical analysis of the microglial marker Iba-1. The mRNA and protein levels of pro-inflammatory cytokines (IL-1β, TNFα, IL-6) were detected by real-time RT-PCR and ELISA. The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by Western blot and immunofluorescence staining. The analgesic effect of exogenous recombinant TSG-6 on CCI-induced mechanical allodynia and heat hyperalgesia was observed by behavioral tests. In the in vitro experiments, primary cultured microglia were stimulated with the TLR2 agonist Pam3CSK4, and then co-cultured with BMSCs or recombinant TSG-6. The protein expression of TLR2, MyD88, p-p65 was evaluated by Western blot. The mRNA and protein levels of IL-1β, TNFα, IL-6 were detected by real-time RT-PCR and ELISA. BMSCs were transfected with the TSG-6-specific shRNA and then intrathecally injected into spinal cerebrospinal fluid in vivo or co-cultured with Pam3CSK4-treated primary microglia in vitro to investigate whether TSG-6 participated in the therapeutic effect of BMSCs on CCI-induced neuropathic pain and neuroinflammation. RESULTS We found that CCI-induced mechanical allodynia and heat hyperalgesia were ameliorated by intrathecal injection of BMSCs. Moreover, intrathecal administration of BMSCs inhibited CCI-induced neuroinflammation in spinal cord tissues. The analgesic effect and anti-inflammatory property of BMSCs were attenuated when TSG-6 expression was silenced. We also found that BMSCs inhibited the activation of the TLR2/MyD88/NF-κB pathway in the ipsilateral spinal cord dorsal horn by secreting TSG-6. Meanwhile, we proved that intrathecal injection of exogenous recombinant TSG-6 effectively attenuated CCI-induced neuropathic pain. Furthermore, in vitro experiments showed that BMSCs and TSG-6 downregulated the TLR2/MyD88/NF-κB signaling and reduced the production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in primary microglia treated with the specific TLR2 agonist Pam3CSK4. CONCLUSIONS The present study demonstrated a paracrine mechanism by which intrathecal injection of BMSCs targets the TLR2/MyD88/NF-κB pathway in spinal cord dorsal horn microglia to elicit neuroprotection and sustained neuropathic pain relief via TSG-6 secretion.

中文翻译：

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骨髓间充质干细胞分泌的抗炎蛋白TSG-6通过抑制脊髓小胶质细胞中的TLR2 / MyD88 /NF-κB信号通路来减轻神经性疼痛。

背景技术神经炎症在神经性疼痛的发展和维持中起着至关重要的作用。最近的证据证明，骨髓间充质干细胞（BMSC）可以通过分泌多种生物活性分子（例如TNF-α刺激的基因6蛋白（TSG-6））来抑制神经性疼痛并具有有效的免疫调节和免疫抑制特性。然而，尚不清楚BMSC是否通过分泌TSG-6发挥其对神经性疼痛的镇痛作用。因此，本研究旨在评估BMSCs释放的TSG-6对大鼠慢性收缩性损伤（CCI）所致神经性疼痛的镇痛作用，并探讨了其在体内和体外的潜在机制。方法从大鼠骨髓中分离骨髓间充质干细胞，并通过流式细胞术和功能分化对其进行鉴定。CCI手术后一天，将约5×106个BMSC鞘内注射到脊髓性脑脊髓液中。在CCI手术后的第1、3、5、7、14天进行了行为测试，包括机械性异常性疼痛，热痛觉过敏和运动功能。处理脊髓以进行小胶质标记物Iba-1的免疫组织化学分析。通过实时RT-PCR和ELISA检测促炎细胞因子（IL-1β，TNFα，IL-6）的mRNA和蛋白质水平。通过蛋白质印迹和免疫荧光染色评估TLR2 / MyD88 /NF-κB信号通路的激活。通过行为测试观察到外源重组TSG-6对CCI诱导的机械性异常性疼痛和热痛觉过敏的镇痛作用。在体外实验中，TLR2激动剂Pam3CSK4刺激了原代培养的小胶质细胞，然后与BMSC或重组TSG-6共培养。通过Western印迹评估TLR2，MyD88，p-p65的蛋白表达。实时RT-PCR和ELISA检测IL-1β，TNFα，IL-6的mRNA和蛋白水平。用TSG-6特异的shRNA转染BMSC，然后将其鞘内注射到体内脊髓脊髓液中，或与Pam3CSK4处理的原发性小胶质细胞在体外共培养，以研究TSG-6是否参与了BMSC对CCI诱导的治疗作用神经性疼痛和神经发炎。结果我们发现鞘内注射骨髓间充质干细胞能改善CCI引起的机械性异常性疼痛和热痛觉过敏。此外，鞘内注射BMSCs可抑制脊髓组织中CCI诱导的神经炎症。当TSG-6表达沉默时，BMSCs的镇痛作用和抗炎特性减弱。我们还发现BMSCs通过分泌TSG-6抑制同侧脊髓背角中TLR2 / MyD88 /NF-κB通路的激活。同时，我们证明鞘内注射外源重组TSG-6可有效减轻CCI诱导的神经性疼痛。此外，体外实验表明，BMSCs和TSG-6下调了原发性小胶质细胞的TLR2 / MyD88 /NF-κB信号传导并减少了促炎性细胞因子（如IL-1β，IL-6和TNF-α）的产生。用特定的TLR2激动剂Pam3CSK4处理。