| 王臣健,陈春晓,徐 涛,汤呈宣.阿魏酸钠对大鼠脊髓损伤中NLRP3炎性小体的影响及机制[J].中国脊柱脊髓杂志,2021,(8):739-748. |
| 阿魏酸钠对大鼠脊髓损伤中NLRP3炎性小体的影响及机制 |
| 中文关键词: 脊髓损伤 阿魏酸钠 NLRP3炎性小体 自噬 溶酶体 |
| 中文摘要: |
| 【摘要】 目的：探究阿魏酸钠（SF）对大鼠脊髓损伤（SCI）模型中NLRP3炎性小体的影响及机制。方法：体内研究采用压迫法建立大鼠脊髓损伤模型，雌性SD大鼠随机分成3组（n=12），Sham组不损伤脊髓，SCI+SF组按100mg/kg/d的剂量连续腹腔注射SF给药7d，SCI组给予等量的生理盐水溶液，术后第3天提取脊髓组织蛋白进行Western blot检测NLRP3和激活效应蛋白pro-caspase-1、P20、IL-1β以及自噬蛋白P62、LC3Ⅱ的表达，并对脊髓样本进行免疫荧光染色观察NLRP3变化，术后第7天对脊髓样本进行Nissl染色检测神经元存活情况。体外实验中对大鼠小胶质细胞（正常培养为对照组）采用LPS诱导NLRP3表达（LPS组），加入自噬阻断剂氯喹（CQ）（CQ组及LPS＋CQ组）检测NLRP3激活效应以及溶酶体组织蛋白酶B（Cathepsin B，CTSB）活性，验证自噬与溶酶体功能对NLRP3激活的影响，再通过SF干预（LPS+CQ+SF组）带来的分子水平变化探讨其中的机制。结果：与Sham组相比，SCI组NLRP3表达升高并被激活，pro-caspase-1降解减少，活性产物P20、IL-1β增多，且自噬蛋白p62、LC3Ⅱ均在较高水平（P＜0.05）；与SCI组比较，SCI+SF组NLRP3数量减少，激活减弱，P62、LC3Ⅱ水平下降（P＜0.05），Nissl染色显示前角神经元存活情况改善（P＜0.05）。与之相似，体外实验中，相比对照组，LPS组细胞NLRP3的表达上升（P＜0.05），但激活不显著，而LPS+CQ组NLRP3的水平更高，激活明显，pro-caspase-1降解，活性产物P20、IL-1β增多，此外，P62、LC3Ⅱ的水平异常升高，CTSB活性下降（P＜0.05），而相比LPS+CQ组，LPS+CQ+SF组中P62、LC3Ⅱ的水平出现同步下降，CTSB活性增强（P＜0.05），自噬流恢复。结论：阿魏酸钠通过保护自噬和溶酶体的功能，能够抑制NLRP3炎性小体的产生和激活，从而减轻SCI大鼠神经细胞损伤。 |
Effect and mechanism of sodium ferulate on NLRP3 inflammasomes in spinal cord injury |
| 英文关键词:Spinal cord injury Sodium ferulate NLRP3 inflammasomes Autophagy Lysosomal |
| 英文摘要: |
| 【Abstract】 Objectives: To investigate the effect and mechanism of sodium ferulate(SF) on NLRP3 inflammasomes in the rat model of spinal cord injury(SCI). Methods: In vivo studies, the model of SCI was established by clip compression as previously described, and female SD rats were randomly divided into 3 groups (n=12), spinal cords of the Sham group did not suffer injury, the SCI+SF group was treated with SF by intraperitoneal injection(100mg/kg/d) for 7 days in a row, and the SCI group was given the same amount of saline. The expressions of NLRP3, and its effector protein pro-caspase-1, P20, IL-1, autophagy protein P62, LC3Ⅱ were detected by Western blot. Western blot on the 3rd day after surgery, and immunofluorescence staining for NLRP3 was also performed. Nissl staining was used to measure the survival of neurons on the 7th day after surgery. In vitro experiments, rat microglia cells(normal culture was used as control group) were treated with LPS(LPS group) to induce NLRP3 expression. To verify the effect of autophagy and lysosomal function on NLRP3, activation of NLRP3 and lysosomal Cathepsin B(CTSB) were detected after following addition of autophagy blocker Chloroquine(CQ)(LPS+CQ group). Finally, the molecular changes induced by SF(LPS+CQ+SF group) intervention were analyzed to explore the mechanism. Results: In vivo studies, compared with the Sham group, the NLRP3 inflammasomes in the SCI group were increased and activated, leading to the degradation of pro-caspase-1 and the increased production of P20 and IL-1β. Moreover, autophagy markers of p62 and LC3Ⅱ were both at higher levels(P＜0.05). Compared with the SCI group, the expression and activation of NLRP3 inflammasomes in the SCI+SF group were decreased, and so were the markers of p62 and LC3Ⅱ(P＜0.05). Nissl staining showed that the survival of neurons in anterior horn of spinal cord was improved(P＜0.05). In vitro experiments, the result showed that the NLRP3 inflammasomes of microglia in the LPS group were induced to generated compared with the control group(P＜0.05), but their activation were not significant. However, the level of NLRP3 in the LPS+CQ group was much higher, accompanied by the significant degeneration of pro-caspase-1 and the increased production of P20 and IL-1β. Moreover, the autophagy markers of P62 and LC3Ⅱ were abnormally elevated, due to their inhibited degradation. Also the maturation of the lysosomestic Cathepsin B(CTSB) was decreased(P＜0.05). However, compared with the LPS+CQ group, the levels of P62 and LC3Ⅱ in the LPS+CQ+SF group were declined synchronously and the maturation of CTSB was improved(P＜0.05), autophagy flux showed a tendency to recover. Conclusions: Sodium ferulate reduces the generation and activation of NLRP3 inflammasomes by protecting the function of autophagy and lysosome. Thus, the neuronal damage of SCI rats is alleviated at last. |
| 投稿时间：2020-07-30 修订日期：2021-07-07 |
| DOI： |
| 基金项目:温州市医药卫生科学研究项目（2016B26）, 白求恩医学科学研究基金（KX038FN） |
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