摘要
目的探究一种双胍二体(LCC-12)对小胶质细胞表面糖蛋白CD44介导的核因子(NF-κB)通路以及对脊髓损伤大鼠病理生理的影响。方法实验于2023年10月至2024年6月进行,用LCC-12体外干预脂多糖(LPS)诱导的小胶质细胞BV-2(LCC-12组),检测CD44、NF-κB等蛋白的表达水平,与单纯用LPS诱导的小胶质细胞(LPS组)进行对比。同时采用来自新疆医科大学实验动物中心的大鼠构建脊髓损伤模型,单纯随机将其分为假手术组(脊髓完好+生理盐水)、对照组(脊髓损伤+生理盐水)和实验组(脊髓损伤+LCC-12),取术后1、3、5、7 d的脊髓组织进行苏木精-伊红和尼氏染色,观察脊髓损伤情况,两组间采用独立样本t检验,多组间比较采用单因素方差分析。结果体外实验显示,LCC-12组白细胞介素(IL)-6的分泌水平低于LPS组[(189.35±8.03)pg/ml比(219.05±5.77)pg/ml,t=3.46,P<0.05],CD44、NF-κB和IL-1蛋白表达水平低于LPS组(1.04±0.06比1.41±0.06、1.12±0.04比1.52±0.06、0.84±0.04比1.11±0.03,t=3.46、4.71、3.67,P<0.05),IκB的表达量高于LPS组(1.41±0.04比0.62±0.03,t=6.93,P<0.05),CD44v6的表达水平低于LPS组(1.73±0.08比4.75±0.13,t=5.93,P<0.05)。动物实验结果显示,实验组术后1 d、7 d和14 d的BBB评分均高于对照组[(2.3±0.9)分比(1.5±0.8)分、(6.1±1.6)分比(4.2±1.5)分、(9.2±2.3)分比(5.3±2.0)分,t=2.10、2.74、4.05,P<0.05];实验组脊髓空洞面积占比和瘢痕组织低于对照组[(10.64±1.34)%比(16.66±1.13)%,t=3.44,P<0.05];尼氏结果显示,实验组神经元存活量高于对照组[每高倍镜视野下,(13.00±0.82)个比(4.00±1.29)个,t=14.42,P<0.05];Western blot结果显示,实验组的JAK2(1.63±0.11比1.51±0.21、1.72±0.03比1.49±0.06、1.79±0.04比1.67±0.06、2.23±0.11比1.75±0.10,t=2.28、3.24、2.74、2.98,P<0.05)、STAT1(2.23±0.07比0.71±0.06、2.25±0.09比2.07±0.10、2.03±0.07比1.58±0.08、1.62±0.07比1.49±0.03,t=7.24、2.54、3.85、2.67,P<0.05)和IL-6(0.90±0.15比0.83±0.11、1.07±0.15比0.87±0.22、1.91±0.13比1.33±0.23、2.83±0.17比2.03±0.33,t=2.35、2.46、4.56、4.67,P<0.05)表达水平均低于同期对照组。结论LCC-12可通过CD44介导NF-κB通路抑制小胶质细胞分泌炎性因子,降低脊髓损伤的炎性反应,减少脊髓空洞和神经胶质瘢痕的形成。
Objective To investigate the effects of a biguanide dimer(LCC-12)on CD44-mediated NF-κB pathway in microglial cells and its impact on the pathophysiology of spinal cord-injured rats.Methods The experiment will be conducted from October 2023 to June 2024.In vitro,LCC-12 was administered to LPS-induced BV-2 microglial cells(LCC-12 group),and protein expression levels of CD44 and NF-κB were assessed.Results were compared with those from cells induced solely by LPS(LPS group).In vivo,rats from Xinjiang Medical University were randomly divided into three groups:sham(spinal cord intact+saline),control(SCI+saline),and experimental(SCI+LCC-12).Spinal tissues were collected postoperatively at days 1,3,5,and 7 for H&E and Nissl staining.t-tests were used for comparisons between two groups and ANOVA for multiple groups.Results In vitro experiments demonstrated that the LCC-12 group exhibited significantly lower levels of interleukin(IL)-6 secretion than the LPS group[(189.35±8.03)pg/ml vs.(219.05±5.77)pg/ml,t=3.46,P<0.05].Additionally,protein expression levels of CD44,NF-κB,and IL-1 were reduced in the LCC-12 group(1.04±0.06 vs.1.41±0.06,1.12±0.04 vs.1.52±0.06,0.84±0.04 vs.1.11±0.03,t=3.46,4.71,3.67,P<0.05),and the IκB expression was increased(1.41±0.04 vs.0.62±0.03,t=6.93,P<0.05).The expression level of CD44v6 was also markedly lower in the LCC-12 group(1.73±0.08 vs.4.75±0.13,t=5.93,P<0.05).In vivo,the experimental group showed higher BBB scores post-surgery at days 1,7,and 14[(2.3±0.9)points vs.(1.5±0.8)points,(6.1±1.6)points vs.(4.2±1.5)points,(9.2±2.3)points vs.(5.3±2.0)points,t=2.10,2.74,4.05,P<0.05],smaller syrinx area percentage and scar tissue formation[(10.64±1.34)%vs.(16.66±1.13)%,t=3.44,P<0.05],and increased neuronal survival[(13.00±0.82)cells vs.(4.00±1.29)cells per high-power field,t=14.42,P<0.05).Western blotting analysis revealed decreased expression levels of JAK2,STAT1,and IL-6 in the experimental group compared to controls(JAK2:1.63±0.11 vs.1.51±0.21,1.72±0.03 vs.1.49±0.06,1.79±0.04 vs.1.67±0.06,2.23±0.11 vs.1.75±0.10,t=2.28,3.24,2.74,2.98,P<0.05;STAT1:2.23±0.07 vs.0.71±0.06,2.25±0.09 vs.2.07±0.10,2.03±0.07 vs.1.58±0.08,1.62±0.07 vs.1.49±0.03,t=7.24,2.54,3.85,2.67,P<0.05;IL-6:0.90±0.15 vs.0.83±0.11,1.07±0.15 vs.0.87±0.22,1.91±0.13 vs.1.33±0.23,2.83±0.17 vs.2.03±0.33,t=2.35,2.46,4.56,4.67,P<0.05).Conclusion LCC-12 inhibits microglial inflammation via the CD44-mediated NF-κB pathway,reducing SCI inflammation and glial scar formation.
作者
孙广旭
高书涛
胡宇坤
冯敬昇
肖扬
盛伟斌
Sun Guangxu;Gao Shutao;Hu Yukun;Feng Jingsheng;Xiao Yang;Sheng Weibin(Department of Spine Surgery,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)
出处
《中华实验外科杂志》
2025年第4期688-691,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(82360257)
新疆维吾尔自治区自然科学基金重点项目(2021D01D18)
青年科研基金(2023YFY-QKMS-06)
天山英才项目(2023TSYCLJ0031)。
