氟伐他汀通过抑制TRPC6保护嘌呤霉素氨基核苷诱导的足细胞损伤

doi:10.5246/jcps.2022.05.031

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (5): 360-368.DOI: 10.5246/jcps.2022.05.031

氟伐他汀通过抑制TRPC6保护嘌呤霉素氨基核苷诱导的足细胞损伤

高慧¹, 张云静², 林静³, 孙希锋²^,^*()

1. 淄博市中心医院泌尿外科, 山东淄博 255036
2. 淄博市中心医院肾内科, 山东淄博 255036
3. 陕西省人民医院心内科, 陕西西安 710068

收稿日期:2021-11-12 修回日期:2021-11-30 接受日期:2021-12-21 出版日期:2022-06-02 发布日期:2022-06-02
通讯作者: 孙希锋
作者简介:
+ Tel.: +86-533-2369195, E-mail: sxfgh-1976@163.com
基金资助:
National Natural Science Foundation of China (Grant No. 81400333).

Fluvastatin protects against puromycin aminonucleoside-induced podocyte injury by inhibiting TRPC6

Hui Gao¹, Yunjing Zhang², Jing Lin³, Xifeng Sun²^,^*()

1 Department of Urology, Zibo Central Hospital, Zibo 255036, China
2 Department of Nephrology, Zibo Central Hospital, Zibo 255036, China
3 Department of Cardiology, Shaanxi Provincial People’s Hospital, Xian 710068, China

Received:2021-11-12 Revised:2021-11-30 Accepted:2021-12-21 Online:2022-06-02 Published:2022-06-02
Contact: Xifeng Sun

摘要/Abstract

摘要：

在本研究中, 我们观察了嘌呤霉素氨基核苷(PAN)对小鼠足细胞瞬时受体电位阳离子通道蛋白6(TRPC6)表达和分布的影响, 并进一步研究了氟伐他汀对TRPC6所致足细胞损伤的保护机制。PAN在不同剂量和不同时间点处理足细胞, 在mRNA和蛋白质水平上评估TRPC6的表达, 免疫荧光法观察TRPC6的分布。然后再将体外培养的足细胞分为4组, 分别检测TRPC6在mRNA和蛋白水平表达的变化, 用激光共聚焦显微镜测定足细胞内钙浓度的变化, 应用Millicell PCF评估BSA的细胞通透性。暴露于PAN后, TRPC6在mRNA和蛋白质水平的表达以剂量和时间依赖性方式增加。免疫荧光显示, 将PAN应用于足细胞后, TRPC6的表达强度显著增加, 并且TRPC6的分布发生改变。在氟伐他汀干预下, PAN诱导的TRPC6上调得以显著逆转。OAG激活TRPC6后, PAN组细胞内钙峰值与基础钙值的比值显著升高, 而在氟伐他汀作用下则明显逆转。PAN处理48 h后足细胞通透性显著增加, 而氟伐他汀干预后上述情况得到有效改善。TRPC6的分布和表达变化与PAN诱导足细胞损伤有关, 氟伐他汀可通过下调TRPC6对足细胞发挥保护作用。

关键词: 瞬时受体单位阳离子通道蛋白6, 足细胞, 嘌呤霉素氨基核苷, 氟伐他汀, 钙离子

Abstract:

In the present study, we aimed to investigate the effect of puromycin aminonucleoside (PAN) on the expression and distribution of transient receptor potential canonical 6 (TRPC6) of murine podocytes and further study the protective mechanism of fluvastatin on podocyte injury by TRPC6 in vitro. Podocytes were treated by PAN at different doses and at different time points. The expressions of TRPC6 at mRNA and protein levels were assessed. An immunofluorescent assay was used to observe the distribution of TRPC6. Cultured podocytes were then divided into four groups. The expressions of TRPC6 at mRNA and protein levels were measured. The intracellular calcium concentration of podocytes was measured with a laser-scanning confocal microscope. The paracellular permeability to BSA was evaluated using Millicell-PCF Inserts. The expressions of TRPC6 at mRNA and protein levels were increased in a dose and time-dependent manner after exposure to PAN. Immunofluorescence showed that the expression intensity of TRPC6 was significantly increased, and the distribution of TRPC6 was changed after PAN was applied to podocytes. With fluvastatin intervention, PAN-induced up-regulation of TRPC6 was significantly reversed. The ratio of the peak value of intracellular calcium to the basic calcium value in the PAN group was significantly higher after TRPC6 was activated by OAG, while it is obviously reversed under the action of fluvastatin. The podocyte permeability was significantly increased after 48 h of PAN treatment, while the above situation was effectively improved after fluvastatin intervention. The changes of distribution and expression of TRPC6 were related to podocyte injury induced by PAN. Fluvastatin could exert its protective effects on podocytes by down-regulating TRPC6.

Key words: TRPC6, Podocyte, Puromycin aminonucleoside, Fluvastatin, Calcium

Supporting:

高慧, 张云静, 林静, 孙希锋. 氟伐他汀通过抑制TRPC6保护嘌呤霉素氨基核苷诱导的足细胞损伤[J]. 中国药学(英文版), 2022, 31(5): 360-368.

Hui Gao, Yunjing Zhang, Jing Lin, Xifeng Sun. Fluvastatin protects against puromycin aminonucleoside-induced podocyte injury by inhibiting TRPC6[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(5): 360-368.

图/表 6

Figure 1. Changes of TRPC6 mRNA and protein under the stimulation of different doses of PAN. Podocytes were treated with PAN 10, 30, 50 and 70 mg/L for 24 h, then the expressions of TRPC6 at mRNA and protein levels were detected. x ± s, n = 3, *P < 0.05, **P < 0.01 compared with the control group. (A) RT-PCR; (B) Western; (C) was the semi-quantitative result of (A) and (B).

Figure 2. Changes of TRPC6 mRNA and protein under the stimulation of PAN at different time points. Podocytes were treated with PAN 50 mg/L for 6, 12, 24 and 48 h, then the expressions of TRPC6 at mRNA and protein levels were detected. x ± s, n = 3, *P < 0.05, **P < 0.01 compared with the control group. A: RT-PCR; B: Western; C was the semi-quantitative result of A and B.

Figure 3. Changes in TRPC6 distribution under the stimulation of PAN at different time points. Under the action of PAN 50 mg/L for 24 h and 48 h, the expression of TRPC6 on podocytes was gradually increased, and its distribution on the cell membrane changed from a dot-like distribution to a coarse-grained shape.

Figure 4. Changes of TRPC6 mRNA and protein in each group of cells. Podocytes were treated with PAN 30 mg/L, PAN 30 mg/L + Fluvastatin 1 μmolL, and Fiuvastatin1 μmol/L, and then the expressions of TRPC6 at mRNA and protein levels were detected after 24 h. x ± s, n = 3, *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the PAN group. A: RT-PCR; B: Western; C was the semi-quantitative result of A and B.

Figure 5. Changes in calcium ion concentration in each group of cells. After OAG 100 μmol/L treatment, the intracellular calcium ion concentration was observed, and calcium ion curves were drawn. x ± s, n = 3, *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the PAN group.

Figure 6. Changes in cell monolayer permeability in each group. Podocytes were treated with PAN 30 mg/L, PAN 30 mg/L + Fluvastatin 1 μmol/L, and Fiuvastatin 1 μmol/L , the podocyte permeability was detected after 48 h. x ± s, n = 3, *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with the PAN group.

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氟伐他汀通过抑制TRPC6保护嘌呤霉素氨基核苷诱导的足细胞损伤

Fluvastatin protects against puromycin aminonucleoside-induced podocyte injury by inhibiting TRPC6

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