Abstract:

Objective　The mouse models of glomerular podocyte injury induced by puromycin aminonucleoside (PAN) were established. After the intervention with glycyrrhizin, the models were observed for 8, 24, and 48 h. To explore the effects of glycyrrhizin on the expression and distribution of Nephrin and α-SMA and its inhibition of podocyte injury. Methods　The experiment was was from June 2024 to May 2025. The experiment included a control group, a PAN group, and a glycyrrhizin group. The control group was cultured in the RPMI 1640 medium containing 0.02% DMSO. On the basis of the control group, 50 mg/L PAN was added to take effect on the podocytes in the PAN group. On the basis of the control group, 50 mg/L PAN and 500 mg/L glycyrrhizin were added to take effect on the podocytes in the glycyrrhizin group. The podocytes' morphology was observed and photographed by an inverted microscope. The expressions of Nephrin and α-SMA mRNA were detected by the real-time fluorescent quantitative polymerase chain reaction (PCR). The expressions of Nephrin and α-SMA were detected by the Western blot. The distribution of α-SMA was observed by the immunofluorescence. LSD-t test and analysis of variance were used as the statistical methods. Results　⑴ After the intervention, all the groups were observed for 8, 24, and 48 h by a microscope. In the control group, the podocytes were clear in shape, plump in cell body, and tight in intercellular connection. In the PAN group, the foot processes retracted; some even disappeared; the intercellular connections were loose. In the glycyrrhizin group, the morphology and structure of the podocytes gradually became clearer. The intercellular connections were more closely formed compared to the PAN group, and the cell bodies were larger than those in the PAN group. ⑵ The results of real-time fluorescent quantitative PCR showed that: the expressions of Nephrin and α-SMA mRNA in the control group were less. In the PAN group, the expression of Nephrin mRNA gradually decreased with the extension of PAN injury time, and decreased to the lowest level at 48 h (P<0.01); while the expression of α-SMA mRNA gradually increased with the extension of PAN injury time, and reached the highest level at 48 h (P<0.01). The expressions of Nephrin mRNA and α-SMA mRNA in the glycyrrhizin group were significantly higher than those in the PAN group, with statistical differences (both P<0.01). ⑶ The Western blot results showed that: in the control group, the expression levels of Nephrin and α-SMA proteins in the podocytes were less. In the PAN group, the expression of Nephrin gradually decreased and the expression of α-SMA gradually increased with the extension of PAN injury time; the expression of Nephrin was the lowest, and the expression of α-SMA the highest (both P<0.01) at 48 h. After intervention, the expressions of nephrin and α-SMA in the glycyrrhizin group were better than those in the PAN group, with statistical differences (both P<0.05). ⑷ The immunofluorescence results showed that: the distribution of α-SMA protein in cell membrane and cytoplasm increased significantly at 24 and 48 h in the PAN group, and the fluorescence intensity increased. The distribution of α-SMA protein in the glycyrrhizin group was better than that in the PAN group. Conclusions　PAN influences the expression and distribution of podocyte marker proteins (Nephrin and a-SMA), resulting in podocyte damage and subsequently proteinuria. Glycyrrhizin can stabilize the expression of these proteins, prevents podocyte damage, and maintains podocyte structural and functional stability.

Key words:

Podocytes, Glycyrrhizin, Puromycin, Nephrin, α-SMA

摘要：

目的　通过嘌呤霉素氨基核苷（puromycin aminonucleoside，PAN）构建小鼠肾小球足细胞损伤模型，甘草甜素干预后观察8、24和48 h，探讨甘草甜素调控Nephrin及a-SMA的表达及分布以抑制足细胞损伤的机制。方法　实验时间为2024年6月至2025年5月，设计对照组、PAN组及甘草甜素组。对照组：用含0.02%二甲基亚砜（DMSO）的RPMI1640培养液培养；PAN组：在对照组的基础上+50 mg/L的PAN进行培养；甘草甜素组：在对照组的基础上+50 mg/L PAN+500 mg/L甘草甜素进行培养。通过倒置显微镜观察足细胞的形态并拍照；实时荧光定量聚合酶链式反应（PCR）检测Nephrin、a-SMA mRNA表达；Western blot检测Nephrin、a-SMA表达，免疫荧光观察a-SMA的分布。统计学方法采用LSD-t检验、方差分析。结果　⑴干预后显微镜观察8、24和48 h。对照组：足细胞形态清晰，胞体饱满，细胞间连接紧密。PAN组：足突回缩，个别甚至消失，细胞间连接松散；甘草甜素组：足细胞形态结构逐渐清晰，细胞间连接较PAN组紧密，胞体大于PAN组。⑵实时荧光定量PCR结果。对照组Nephrin、α-SMA mRNA表达量均较少。PAN组：随PAN损伤时间延长，Nephrin mRNA表达逐渐减弱，48 h降至最低水平（P<0.01）；而α-SMA mRNA表达逐渐增强，48 h达到最高峰（P<0.01）。甘草甜素组：Nephrin mRNA及α-SMA mRNA表达较PAN组改变，差异有统计学意义（P<0.01）。⑶Western Blot结果。对照组：足细胞内标志蛋白Nephrin、α-SMA蛋白表达量均较少；PAN组：随PAN损伤时间延长，Nephrin表达逐渐减弱，α-SMA表达逐渐增强，48 h时Nephrin表达最弱、α-SMA表达最强（均P<0.01）；甘草甜素组：干预后其Nephrin表达和α-SMA表达均较PAN组明显好转，差异有统计学意义（P<0.05）。⑷免疫荧光结果。PAN组α-SMA蛋白分布在24、48 h细胞膜和细胞质中在明显增加，荧光强度增加；甘草甜素组α-SMA蛋白分布较PAN组荧光强度明显改善。结论　PAN通过影响足细胞标志蛋白Nephrin、a-SMA的表达及分布，导致足细胞损伤，继而发生蛋白尿，甘草甜素可使上述蛋白表达趋于稳定，从而发挥保护足细胞损伤作用，有助于维持足细胞结构及功能的稳定。

关键词:

足细胞, 甘草甜素, 嘌呤霉素氨基核苷, Nephrin, α-SMA