葛根来源外泌体样纳米囊泡对D-半乳糖诱导L929小鼠成纤维细胞衰老的改善作用及其机制

doi:10.13481/j.1671-587X.20260302

摘要/Abstract

摘要：

目的探讨葛根来源外泌体样纳米囊泡（PL-ELNs）对D-半乳糖（D-gal）诱导的L929小鼠成纤维细胞衰老的影响，阐明其可能的作用机制。方法采用差速离心结合蔗糖密度梯度离心从葛根中提取PL-ELNs，采用透射电镜（TEM）、纳米颗粒跟踪分析（NTA）和试剂盒分别对其进行形态观察、粒径鉴定和蛋白浓度定量。将L929细胞分为对照组、D-gal组和4、8及16 mg·L^-1 PL-ELNs+D-gal组，采用PKH26标记的PL-ELNs处理L929细胞，观察L929细胞对PL-ELNs的摄取情况。使用D-gal诱导构建L929细胞衰老模型，采用细胞计数试剂盒8（CCK-8）法筛选D-gal和PL-ELNs最佳作用浓度并检测各组细胞活性，β-半乳糖苷酶（SA-β-gal）染色观察各组L929细胞染色情况并计算SA-β-gal染色阳性细胞百分率。采用5-乙炔基-2'-脱氧尿苷（EdU）染色检测各组L929细胞增殖活性，流式细胞术检测各组细胞内活性氧（ROS）水平和细胞凋亡率，Western blotting法检测各组L929细胞中Ⅰ型胶原蛋白（ColⅠ）、Ⅲ型胶原蛋白（ColⅢ）、P21和P16蛋白表达水平。结果从葛根中分离出茶托样形态、中位粒径为142.3 nm的PL-ELNs，并可被L929细胞有效摄取。20 g·L^-1 D-gal可以成功诱导L929细胞衰老。CCK-8法，与对照组比较，D-gal组L929细胞活性明显降低（P<0.01）；与D-gal组比较，4、8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞活性明显升高（P<0.01）。SA-β-gal染色，与对照组比较，D-gal组L929细胞中SA-β-gal染色阳性细胞百分率明显升高（P<0.01）；与D-gal组比较，4、8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞中SA-β-gal染色阳性细胞百分率均明显降低（P<0.05或P<0.01）。Western blotting法，与对照组比较，D-gal组L929细胞中P21和P16蛋白表达水平明显升高（P<0.01）；与D-gal组比较，8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞中P21和P16蛋白表达水平明显降低（P<0.05或P<0.01）。EdU染色，与对照组比较，D-gal组L929细胞增殖活性明显降低（P<0.01）；与D-gal组比较，4、8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞增殖活性均明显升高（P<0.05或P<0.01）。流式细胞术，与对照组比较，D-gal组L929细胞中ROS水平明显升高（P<0.01）；与D-gal组比较，8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞中ROS水平明显降低（P<0.05或P<0.01）。与对照组比较，D-gal组L929细胞凋亡率明显升高（P<0.01），4、8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞凋亡率均明显降低（P<0.01）。Western blotting法，与对照组比较，D-gal组L929细胞中ColⅠ和ColⅢ蛋白表达水平明显降低（P<0.01）；与D-gal组比较，8和16 mg·L^-1 PL-ELNs+D-gal组L929细胞中ColⅠ和ColⅢ蛋白表达水平均明显升高（P<0.05或P<0.01）。结论 PL-ELNs可改善D-gal诱导的L929细胞衰老，其机制可能与提高衰老细胞的增殖活性、减少细胞凋亡、降低细胞中ROS水平及P21和P16蛋白表达水平有关。

关键词: 葛根, 外泌体, 纳米囊泡, 细胞衰老, D-半乳糖, L929细胞

Abstract:

Objective To discuss the effect of Pueraria lobata-derived exosome-like nanovesicles （PL-ELNs） on senescence of L929 mouse fibroblasts induced by D-galactose （D-gal）， and to clarify the possible mechanism of action. Methods The PL-ELNs were extracted from Pueraria lobata by differential centrifugation combined with sucrose density gradient centrifugation. Transmission electron microscope （TEM） was used to observe the morphology of PL-ELNs； nanoparticle tracking analysis （NTA） was used to identify the particle size of PL-ELNs； kit method was used to quantify the protein concentration of PL-ELNs. The L929 cells were divided into control group， D-gal group， and 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups. The PKH26-labeled PL-ELNs were used to treat the L929 cells， and the uptake of PL-ELNs by L929 cells was observed. D-gal was used to establish the senescence model of L929 cells. CCK-8 method was used to screen the optimal concentrations of D-gal and PL-ELNs and to detect the activities of the cells in various groups； SA-β-gal staining was used to observe the staining of L929 cells in various groups and to calculate the percentage of SA-β-gal staining positive cells. EdU staining was used to detect the percentages of EdU positive cells in L929 cells in various groups； flow cytometry was used to detect the intracellular reactive oxygen species （ROS） levels and apoptotic rates of L929 cells in various groups； Western blotting method was used to detect the expression levels of collagen type Ⅰ （ColⅠ）， collagen type Ⅲ （ColⅢ）， P21 and P16 proteins in L929 cells in various groups. Results The PL-ELNs with a saucer-like morphology and a median particle size of 142.3 nm were isolated from Pueraria lobata， and could be effectively taken up by L929 cells. D-gal at 20 g·L?1 successfully induced senescence of L929 cells. The CCK-8 assay results showed that compared with control group， the activity of L929 cells in D-gal group was significantly decreased （P<0.01）； compared with D-gal group， the activities of L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.01）. The SA-β-gal staining results showed that compared with control group， the percentage of SA-β-gal staining positive L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the percentages of SA-β-gal staining positive L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. The Western blotting results showed that compared with control group， the expression levels of P21 and P16 proteins in L929 cells in D-gal group were significantly increased （P<0.01）； compared with D-gal group， the expression levels of P21 and P16 proteins in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. The EdU staining results showed that compared with control group， the percentage of EdU positive L929 cells in D-gal group was significantly decreased （P<0.01）； compared with D-gal group， the percentages of EdU positive L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.05 or P<0.01）. The flow cytometry results showed that compared with control group， the ROS levels in L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the ROS levels in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.05 or P<0.01）. Compared with control group， the apoptotic rate of L929 cells in D-gal group was significantly increased （P<0.01）； compared with D-gal group， the apoptotic rates of L929 cells in 4， 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of ColⅠ and ColⅢ proteins in L929 cells in D-gal group were significantly decreased （P<0.01）； compared with D-gal group， the expression levels of ColⅠ and ColⅢ proteins in L929 cells in 8 and 16 mg·L^-1 PL-ELNs+D-gal groups were significantly increased （P<0.05 or P<0.01）. Conclusion PL-ELNs can ameliorate D-gal-induced senescence of L929 cells， and the mechanism may be related to increasing the proliferation activity of senescent cells， reducing apoptosis， and decreasing the ROS level and the expression levels of P21 and P16 proteins in the cells.

Key words: Pueraria lobata, Exosome, Nanovesicle, Cell senescence, D-galactose, L929 cells

中图分类号:

R711.4

向春蓉,何勇,蒋诺,黄首琪,洪莉. 葛根来源外泌体样纳米囊泡对D-半乳糖诱导L929小鼠成纤维细胞衰老的改善作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(3): 590-601.

Chunrong XIANG,Yong HE,Nuo JIANG,Shouqi HUANG,Li HONG. Ameliorative effect of Pueraria lobata-derived exosome-like nanovesicles on D-galactose-induced senescence in L929 mouse fibroblasts and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 590-601.

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