刺槐提取物通过稳定肌动蛋白骨架保护小鼠足细胞

doi:10.5246/jcps.2021.03.018

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (3): 230-238.DOI: 10.5246/jcps.2021.03.018

刺槐提取物通过稳定肌动蛋白骨架保护小鼠足细胞

罗璇¹, 杜清¹, 赵海燕¹, 范文海¹, 高欣², 高霞³^,^*()

1. 甘肃省人民医院儿科, 甘肃兰州 730000
2. 甘肃省人民医院 ICU, 甘肃兰州 730000
3. 广州市妇女儿童医疗中心肾脏科, 广东广州 510623

收稿日期:2020-07-29 修回日期:2020-08-12 接受日期:2020-11-06 出版日期:2021-03-29 发布日期:2021-03-29
通讯作者: 高霞
作者简介:
+ Tel.: +86-13711025095, E-mail: gaoxiagz@vip.163.com
基金资助:
The National Natural Science Foundation of China (Grant No. 81660130) and the Natural Science Foundation of Gansu Province, China (Grant No. 18JR3RA045).

Extractum trametes robiniophila murr protects murine podocyte via actin cytoskeleton stabilization

Xuan Luo¹, Qing Du¹, Haiyan Zhao¹, Wenhai Fan¹, Xin Gao², Xia Gao³^,^*()

1 Department of Pediatric, People’s Hospital of Gansu province, Lanzhou, Gansu 730000, China
2 Department of Intensive care center, People’s Hospital of Gansu province, Lanzhou, Gansu 730000, China
3 Nephrology department, Guangzhou Women and Children’s Medical Center, Guangzhou city 510623, China

Received:2020-07-29 Revised:2020-08-12 Accepted:2020-11-06 Online:2021-03-29 Published:2021-03-29
Contact: Xia Gao

摘要/Abstract

摘要：

槐耳浸膏 (Extractum trametes robiniophila murr, ETRM)是一种真菌的提取物, 最近的研究表明, ETRM能有效地改善阿霉素(ADR)诱导的肾病大鼠足细胞足突的清除。在本研究中, 我们探讨了槐耳浸膏对足细胞骨架重排及足细胞固有分子α-actinin-4、nephrin的调控特点。首先进行分组: 槐耳浸膏组: 槐耳浸膏 (10 mg/mL)＋阿霉素 (0.5 μmol/L); 地塞米松组: 地塞米松 (1 μmol/L) ＋阿霉素 (0.5 μmol/L); 阿霉素组: 阿霉素 (0.5 μmol/L); 正常足细胞组。采用invitrogen?鬼笔环肽直接荧光标记各组足细胞F-actin后, 在激光共聚焦显微镜下观察细胞骨架重排情况。Real-time PCR和Western blott分析各组足细胞α-actinin-4、nephrin的表达变化。未经药物预处理的足细胞在阿霉素干预后, 骨架发生显著重排。槐耳浸膏预处理的足细胞显示出与地塞米松预处理组相似的稳定足细胞骨架, 减轻重排发生的能力。Real-time PCR和Western blott均提示与正常足细胞相比, 槐耳浸膏预处理后足细胞α-actinin-4表达量显著低于阿霉素组, 与正常足细胞组无显著差异。但槐耳浸膏预处理不能缓解阿霉素引起的nephrin表达改变。地塞米松预处理组α-actinin-4和nephrin表达水平均显著低于阿霉素组。槐耳浸膏能够减轻阿霉素诱导的足细胞骨架重排, 其作用机制可能与调控α-actinin-4的表达有关。

关键词: 肌动蛋白重组, 槐耳浸膏, 足细胞, 阿霉素

Abstract:

The Chinese medicine Extractum trametes robiniophila murr (ETRM) is the extract of a type of fungus. Recent studies have suggested that ETRM efficiently improves the effacement of podocyte foot processes in adriamycin (ADR)-induced nephrotic rats. In the present study, we aimed to assess whether ETRM modulated the actin rearrangements of podocytes and involved signaling molecules, including α-actinin-4 and nephrin. Podocytes were treated with ADR (0.5 μmol/L), ADR (0.5 μmol/L) + dexamethasone (Dex) (1 μmol/L), ADR (0.5 μmol/L) + ETRM (10 mg/mL). The F-actin in the podocytes was stained by fluorescent phallotoxins and observed by confocal microscopy. The expression levels of α-actinin-4 and nephrin were tested by real-time PCR and Western blotting analysis. The administration of ETRM could significantly prevent ADR-treated podocytes from actin rearrangement. Both ETRM and Dex could stabilize podocyte actin cytoskeletons. Moreover, α-actinin-4 might act as a potential target for ETRM functionality in podocyte actin rearrangements. However, pretreatment with ETRM could not inhibit the up-regulation of nephrin as a result of ADR treatment. ETRM could improve the cytoskeleton stability in ADR-induced actin rearrangement of podocytes via regulating α-actinin-4 expression.

Key words: Actin rearrangements, Extractum trametes robiniophila murr, Foot processes, Adriamycin

Supporting:

罗璇, 杜清, 赵海燕, 范文海, 高欣, 高霞. 刺槐提取物通过稳定肌动蛋白骨架保护小鼠足细胞[J]. 中国药学(英文版), 2021, 30(3): 230-238.

Xuan Luo, Qing Du, Haiyan Zhao, Wenhai Fan, Xin Gao, Xia Gao. Extractum trametes robiniophila murr protects murine podocyte via actin cytoskeleton stabilization[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(3): 230-238.

图/表 3

Figure 1. ETRM could protect podocytes from ADR-induced actin cytoskeleton rearrangements. (A) Phalloidin-fluorescence labeling of F-actin showed podocyte stress fibers. Confocal microcopy showed that podocyte stress fibers became despoliated after ADR administration. Podocyte cytoskeleton showed well organization in either Dex or ETRM pretreatment group. (B) The percentage of stress fibers in podocytes demonstrated that ADR treatment could significantly decrease podocytic stress fibers. (C) MFI of F-actin under confocal microscopy further indicated that ADR treatment could directly decrease podocyte F-actin content. (D) The stress-fiber percentage in ETRM-pretreated podocytes was significantly improved after ADR administration for 24 h compared with cells without ETRM pretreatment. (E) ETRM significantly increased the F-actin content in podocytes after ADR administration. *P < 0.05, **P < 0.01 (representative of three replicate experiments).

Figure 2. α-Actinin-4 is involved in ETRM-related actin cytoskeleton stabilization. (A) Real-time PCR data showed that α-actinin-4 mRNA in podocytes pretreated with ETRM or Dex for 1 h was not significantly changed after ADR administration. (B) Western blotting analysis also confirmed real-time PCR results. (C) Relative α-actinin-4/GAPDH level *P < 0.05. (Representative of three replicate experiments).

Figure 3. ETRM fails to modulate nephrin expression in ADR-treated podocytes. (A) Real-time PCR data showed that ETRM pretreatment could not inhibit the upregulation of nephrin as result of ADR treatment. (B) Western blotting analysis also confirmed real-time PCR results. (C) relative nephrin/GAPDH level, *P < 0.05, **P < 0.01. (Representative of three replicate experiments).

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刺槐提取物通过稳定肌动蛋白骨架保护小鼠足细胞

Extractum trametes robiniophila murr protects murine podocyte via actin cytoskeleton stabilization

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