Effects of Bone Marrow Mesenchymal Stem Cell Transplantation on Neuropathic Pain in Rats with Spinal Cord Injury and Its Action on LPS-Induced Neuronal Cells
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摘要:
目的 探究骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）治疗对脊髓损伤（spinal cord injury，SCI）神经病理性疼痛大鼠的影响及其作用机制。 方法 将大鼠随机分为假手术（sham）组、骨髓损伤模型（SCI）组、SCI+BMSCs组、SCI+BMSCs+LY294002组（n=15）。利用Sprague-Dawley大鼠诱导SCI模型，并对SCI大鼠损伤脊髓中注射BMSCs和PI3K抑制剂LY294002。测定大鼠（basso-beattie-bresnahan，BBB）评分、爪机械缩回阈值（paw mechanical withdrawal threshold，pMWT）和热刺激下的爪热缩回潜伏期（paw thermal withdrawal latency，pTWL）。苏木精-伊红染色、免疫荧光染色、酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）和Western Blot实验探讨BMSCs移植对SCI大鼠的作用。利用脂多糖（lipopolysaccharide，LPS）诱导大鼠脊髓神经元细胞（rat spinal cord neurons，RSCN），并将其与BMSCs及其外泌体共培养，通过Annexin V-FITC/PI试剂盒、ELISA和Western Blot实验探讨BMSCs及其外泌体对RSCN的作用。 结果 SCI造模后第5天大鼠BBB评分≤5，且第20天BBB评分<10分，HE染色脊髓组织结构疏松、形成大量空泡、神经元细胞萎缩即认为SCI模型成功。与sham组比较，SCI组BBB评分、pMWT和pTWL值降低（P < 0.001），脊髓中IBA1荧光强度、促炎细胞因子和疼痛相关因子的水平增加（P < 0.001），抑制PI3K/AKT信号通路激活。BMSCs移植通过激活PI3K/AKT通路（P < 0.001），对脊髓损伤大鼠具有保护作用。注射LY294002减弱BMSCs对SCI大鼠损伤脊髓修复作用。LPS诱导RSCN细胞凋亡促炎细胞因子水平（P < 0.001），MSCs或BMSCs-exo与RSCN共培养可激活PI3K/AKT信号通路，减少LPS诱导的细胞凋亡和促炎细胞因子生成（P < 0.05）。 结论 BMSCs通过外泌体激活神经元中的PI3K/AKT信号通路，抑制TNF-α、SP、NE、5-HT水平，促进SCI大鼠功能恢复。 Abstract:Objective To investigate the effects of BMSCs treatment on neuropathic pain in rats with SCI and explore the underlying mechanism. Methods The rats were randomly divided into the following groups (n=15 per group): sham-operated (sham) group, spinal cord injury model (SCI) group, SCI + BMSCs group, and SCI + BMSCs + LY294002 group. An SCI model was established using Sprague-Dawley rats, followed by intraspinal administration of BMSCs and the PI3K inhibitor LY294002 to the injured spinal cord of SCI rats. The BBB score, pMWT, and pTWL values under thermal stimulation were measured. Hematoxylin-eosin staining was used to observe pathological changes in the injured spinal cord of rats. The effects of BMSCs transplantation on SCI rats were explored through hematoxylin-eosin staining, immunofluorescence staining, ELISA, and Western Blot experiments. RSCN were induced using LPS and co-cultured with BMSCs and their exosomes. The effects of BMSCs and their exosomes on RSCN were investigated through Annexin V-FITC/PI kit, ELISA, and Western Blot assays. Results The SCI model was considered successfully established when the following criteria were met on post-operative day 5: BBB locomotor score ≤5, accompanied by a BBB score <10 on day 20, along with histopathological evidence of spinal cord tissue loosening, extensive vacuolation, and neuronal atrophy observed via HE staining. Compared with the sham group, the SCI group exhibited significantly lower BBB scores, pMWT, and pTWL values (P < 0.001). Concurrently, increased immunofluorescence intensity of IBA1, elevated levels of pro-inflammatory cytokines, and pain-related factors were detected in the spinal cord (P < 0.001). Furthermore, activation of the PI3K/AKT signaling pathway was significantly suppressed. BMSCs transplantation protected SCI rats by activating the PI3K/AKT pathway （P < 0.001）. BMSC-mediated spinal cord repair was attenuated by LY294002 administration. LPS-induced RSCNs showed increased apoptosis and pro-inflammatory cytokine release （P < 0.001）. Co-culture with MSCs or BMSCs-derived exosomes activated the PI3K/AKT signaling pathway, thereby reducing LPS-induced apoptosis and proinflammatory cytokine production （P < 0.05）. Conclusion BMSCs activate the PI3K/AKT signaling pathway in neurons through exosomes, suppressing the levels of TNF-α, SP, NE, and 5-HT, and promoting functional recovery in SCI rats. -
图 1 大鼠BBB评分、pMWT和pTWL值
A：各组大鼠BBB评分；B：大鼠pMWT值比较；C：大鼠pTWL值比较；与sham组相比，***P < 0.001；与SCI组相比，△△P < 0.01，△△△P < 0.001；与SCI+BMSCs组相比，#P < 0.05，##P < 0.01，###P < 0.001。
Figure 1. BBB score，pMWT，and pTWL values in rats
图 4 ELISA试剂盒检测炎性细胞因子和致痛因子水平（$ \bar x \pm s $，n = 6）
A～E：ELISA检测促炎细胞因子和致痛因子的水平；A：TNF-α；B：IL-4；C：SP；D：NE；E：5-HT；***P < 0.001。
Figure 4. Inflammatory cytokines and nociceptive factors detected by ELISA （$ \bar x \pm s $，n = 6）
图 5 PI3K、AKT及其磷酸化蛋白表达（$ \bar x \pm s $，n = 6）
A～E：Western Blot检测PI3K、PI3K-P85、AKT和AKT-PS473蛋白表达及其相对表达分析；***P < 0.001。
Figure 5. Expression of PI3K，AKT，and their phosphorylated proteins （$ \bar x \pm s $，n = 6）
图 6 骨髓间充质干细胞对LPS诱导神经元细胞的作用（$ \bar x \pm s $，n = 3）
A～B：Annexin V-FITC/PI检测细胞凋亡率；C～D：ELISA试剂盒检测TNF-α和IL-4浓度；**P < 0.01，***P < 0.001。
Figure 6. Effects of bone marrow mesenchymal stem cells on LPS-induced neuronal cells （$ \bar x \pm s $，n = 3）
图 7 BMSCs来源外泌体减轻LPS对神经元细胞的作用（$ \bar x \pm s $，n = 3）
A：外泌体扫描电镜观察结果；B：Western Blot检测外泌体标志物Alix和CD63在BMSCs和BMSCs-exo中的表达；C～D：Annexin V-FITC/PI检测细胞凋亡率；E～F：ELISA检测TNF-α和IL-4浓度；G～K：Western Blot检测PI3K/AKT通路蛋白和其磷酸化水平的表达；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 7. BMSC-derived exosomes alleviate the effects of LPS on neuronal cells （$ \bar x \pm s $，n = 3）
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