碳点-普鲁士蓝纳米酶抗氧化应激延缓椎间盘退变

Carbon dot Prussian blue nanoenzyme antioxidative stress delays intervertebral disc degeneration

背景:近年来研究发现氧化应激是椎间盘退变的重要激活机制,具有类酶活性的纳米颗粒在抗氧化应激等领域受到大量关注,但有关其在延缓椎间盘退变方面的探究较少。目的:构建碳点-普鲁士蓝复合纳米颗粒,通过体内外实验验证其清除过量活性氧、调控氧化应激延缓大鼠椎间盘退变的作用。方法:基于水热法,以聚乙烯亚胺为碳源,与柠檬酸、FeCl_(3)·6H_(2)O混合制备碳点,随后在其表面原位合成普鲁士蓝,构建碳点-普鲁士蓝复合纳米颗粒(记为CD-PBs)。①体外实验:提取SD大鼠原代髓核细胞,培养至3代,然后分别加入H_(2)O_(2)溶液(对照组)、CD-PBs+H_(2)O_(2)溶液(实验组)共培养,以单独培养的细胞为空白对照,细胞被处理24 h后,利用荧光探针检测细胞内活性氧水平与线粒体膜电位,实时定量PCR分析细胞Ⅱ型胶原、聚集蛋白聚糖、基质金属蛋白酶3、肿瘤坏死因子αmRNA表达。②体内实验:将30只SD大鼠随机分3组,空白对照组不做任何处理,对照组建立尾椎椎间盘退变模型,实验组将CD-PBs溶液注射至退变椎间盘间隙内,术后8周,病理切片观察椎间盘形态。结果与结论:①体外实验:对照组细胞内活性氧水平高于空白对照组(P<0.05),实验组细胞内活性氧水平低于对照组(P<0.05)。对照组细胞线粒体荧光强度低于空白对照组(P<0.05),实验组细胞线粒体荧光强度高于对照组(P<0.05)。与空白对照组比较,对照组Ⅱ型胶原、聚集蛋白聚糖mRNA表达量下降(P<0.05),基质金属蛋白酶3、肿瘤坏死因子αmRNA表达量升高(P<0.05);与对照组比较,实验组Ⅱ型胶原、聚集蛋白聚糖mRNA表达量升高(P<0.05),基质金属蛋白酶3、肿瘤坏死因子αmRNA表达量下降(P<0.05)。②体内实验:苏木精-伊红染色显示,对照组大鼠椎间盘间隙高度明显降低,纤维环排列紊乱,且髓核内结构破坏,细胞和基质大量丢失;实验组大鼠椎间盘椎间隙高度下降不明显,髓核部分丢失,纤维环结构稍显紊乱,较对照组有明显改善。③结果表明:碳点-普鲁士蓝复合纳米颗粒可通过清除细胞内活性氧、抗氧化应激延缓椎间盘退变。

BACKGROUND:In recent years,oxidative stress has been found to be an important activation mechanism of intervertebral disc degeneration.Nanoparticles with enzyme-like activity have attracted a lot of attention in the study of anti-oxidant stress,but there is little research on delaying intervertebral disc degeneration.OBJECTIVE:To construct carbon dot Prussian blue nanoparticles(PEI-600-Fe C-dots Prussian Blue Nanoparticles,CD-PBs),to verify that CD-PBs can eliminate excessive reactive oxygen species,regulate oxidative stress and delay intervertebral disc degeneration in rats by in vitro and in vivo experiments.METHODS:Based on the hydrothermal method,polyethyleneimine was used as carbon source to prepare carbon dots by mixing with citric acid and FeCl_(3)•6H_(2)O,and then Prussian blue was synthesized in situ on its surface to construct CD-PBs.(1)In vitro experiment:SD rat nucleus pulposus cells were extracted and cultured for three generations.Then,H_(2)O_(2)solution(control group)and CD-PBs+H_(2)O_(2)solution(experimental group)were added for co-culture,and the cells cultured alone were used as blank controls.After the cells were treated for 24 hours,the level of intracellular reactive oxygen species and mitochondrial membrane potential were detected by fluorescent probes.The mRNA expression levels of type II collagen,aggrecan,matrix metalloproteinase 3 and tumor necrosis factorαwere analyzed by real-time quantitative PCR.(2)In vivo experiment:30 SD rats were randomly divided into three groups.The blank control group did not do any treatment.In the control group,a model of coccygeal intervertebral disc degeneration was established.In the experimental group,CD-PBs solution was injected into the degenerated intervertebral disc space.At 8 weeks after operation,the morphology of intervertebral disc was observed by pathological section.RESULTS AND CONCLUSION:(1)In vitro experiments:The intracellular reactive oxygen species level in the control group was higher than that in the blank control group(P<0.05).The level of intracellular reactive oxygen species in the experimental group was lower than that in the control group(P<0.05).The mitochondrial fluorescence intensity of the control group was lower than that of the blank control group(P<0.05).The mitochondrial fluorescence intensity of the experimental group was higher than that of the control group(P<0.05).Compared with the blank control group,the mRNA expression of type II collagen and aggrecan decreased in the control group(P<0.05);the mRNA expression of matrix metalloproteinase 3 and tumor necrosis factorαincreased(P<0.05).Compared with the control group,the mRNA expression of collagen type II and aggrecan in the experimental group increased(P<0.05);the mRNA expression of matrix metalloproteinase 3 and tumor necrosis factorαdecreased(P<0.05).(2)In vivo experiment:Hematoxylin-eosin staining showed that the height of the intervertebral disc space in the control group was significantly reduced;the arrangement of the annulus fibrosus was disordered;the structure of the nucleus pulposus was destroyed,and the cells and matrix were lost in large quantities.In the experimental group,the height of the intervertebral disc space did not decrease significantly;the nucleus pulposus was partially lost,and the structure of the annulus fibrosus was slightly disordered,which was significantly improved compared with the control group.(3)The results showed that CD-PBs could prevent oxidative stress by removing intracellular reactive oxygen species and delay intervertebral disc degeneration.