雷帕霉素诱导自噬抑制大鼠尿道损伤后纤维化的作用机制研究

陈守峰; 张舒超; 李中; 孙巍; 刘贝贝; 杨小淮; 郭园园

doi:10.16766/j.cnki.issn.1674-4152.003017

雷帕霉素诱导自噬抑制大鼠尿道损伤后纤维化的作用机制研究

doi: 10.16766/j.cnki.issn.1674-4152.003017

蚌埠医学院第一附属医院泌尿外科，安徽蚌埠 233004

基金项目:

安徽省高校优秀青年人才支持计划项目重点项目 gxyqZD2019038

安徽省高校自然科学基金重点项目 KJ2021A0752

详细信息

通讯作者:
郭园园，E-mail：guoshaohua111@163.com

中图分类号: R695 R-332
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- 被引次数: 0
出版历程
- 收稿日期: 2022-08-13
- 网络出版日期: 2023-08-26

The mechanism of rapamycin-induced autophagy to inhibit urethral fibrosis after injury in rats

Department of Urology, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China

摘要

摘要: 目的探究雷帕霉素(Rapa)对大鼠尿道狭窄形成和成纤维细胞的影响及其作用机制。方法构建大鼠尿道狭窄模型(SD雄性大鼠，体质量250~300 g)，采用随机数字表法分为阴性对照组、阳性对照组和Rapa组，每组10只。术后14 d取大鼠尿道组织，行HE染色观察尿道组织形态学变化，Masson染色检测胶原变化；免疫组化法观察α平滑肌肌动蛋白(α-SMA)的表达。将HLF-1细胞分为对照组，Rapa组和3-甲基腺嘌呤(3-MA)+Rapa组。应用transwell实验检测细胞迁移和侵袭能力，流式细胞术检测细胞的周期和凋亡情况，Western blotting检测LC3的表达。结果 HE染色发现Rapa组大鼠尿道组织狭窄程度较阳性对照组明显减轻，Masson及免疫组化染色结果显示，阳性对照组和Rapa组大鼠损伤尿道组织中胶原和α-SMA表达均较阴性对照组大鼠增加，但Rapa组胶原和α-SMA表达显著低于阳性对照组。与对照组相比，Rapa组HLF-1细胞迁移、侵袭能力逐渐减弱，呈剂量依赖关系(均P＜0.05)，而加入3-MA干预后，Rapa对HLF-1迁移、侵袭和能力的抑制作用减弱(均P＜0.05)。流式细胞术及Western blotting结果显示，与对照组相比，Rapa组细胞凋亡明显增加(均P＜0.05)，且细胞LC3表达呈剂量依赖性增多(均P＜0.05)。结论雷帕霉素可以缓解大鼠尿道组织损伤后纤维化，并抑制HLF-1细胞迁移和侵袭能力，诱导HLF-1细胞的凋亡，其效应可能是通过诱导细胞自噬来实现的。
- 雷帕霉素 /
- 人胚肺成纤维细胞 /
- 侵袭迁移 /
- 自噬 /
- 尿道狭窄
Abstract: Objective To investigate the effect and mechanism of rapamycin (Rapa) on the formation of urethral stricture and fibroblast in rats. Methods The rat (SD male rats, body weight 250-300 g) urethral stricture model was established and divided into negative control group, positive control group and Rapa+3-Methyladenine (3-MA) group by using the random nuber table method, with 10 rats in eah group. The urethral tissues of rats were collected at 14 d after operation, and the morphological changes of urethral tissues were observed by HE staining, and the changes of collagen were detected by Masson staining. The expression of α-SMA was observed by immunohistochemistry. HLF-1 cells were divided into control group, Rapa group and 3-MA+Rapa group. Transwell assay was applied to detect cell migration and invasion ability, flow cytometry to detect cell cycle and apoptosis, and Western blotting to detect LC3 expression. Results HE staining showed that urethral stricture degree of rats in the Rapa group was significantly reduced compared with that in the positive control group. Masson and immunohistochemical staining showed that collagen and α-SMA expression in the injured urethra tissues were increased in both the positive control group and Rapa group compared with that in the negative control group. However, the expressions of collagen and α-SMA in Rapa group were significantly lower than those in positive control group. Compared with the control group, the migration and invasion ability of HLF-1 cells in Rapa group were gradually decreased in a dose-dependent relationship (all P < 0.05), while the inhibition of Rapa on the migration, invasion ability of HLF-1 cells was decreased after the addition of 3-MA intervention (all P < 0.05). Flow cytometry and Western blotting results showed a significant increase in apoptosis in the Rapa group compared to the control group (all P < 0.05), and a dose-dependent increase in cellular LC3 expression (all P < 0.05). Conclusion Rapamycin can relieve the fibrosis after urethral tissue injury in rats, inhibit the migration and invasion of HLF-1 cells, and induce the apoptosis of HLF-1 cells, which may be achieved by inducing autophagy.
- Rapamycin /
- Human fetal lung fibroblast 1 /
- Invasion and migration /
- Autophagy /
- Urethral stricture

HTML全文

图 1 3组大鼠尿道组织狭窄比较

注：A为HE染色检测NC组(阴性对照组)、PC组(阳性对照组)及Rapa组尿道组织狭窄及上皮细胞增生情况(×100)；B为Massion染色检测3组尿道组织胶原含量(×100)；C为免疫组化检测3组尿道组织中α-SMA的含量(×100)。

Figure 1. Comparison of urethral strictures among the three groups

下载: 全尺寸图片幻灯片

图 2 不同浓度药物对细胞功能的影响

注：A示transwell检测不同浓度Rapa对细胞迁移及侵袭能力的影响(×50)；B示流式细胞术检测不同浓度Rapa对细胞周期和凋亡的影响。

Figure 2. Effects of different concentrations of drugs on cell function

下载: 全尺寸图片幻灯片

图 3 不同药物对细胞功能的影响

注：A示transwell检测加入3-MA对Rapa组细胞迁移及侵袭能力的影响(×50)；B示Western blotting检测不同浓度Rapa组细胞中LC3蛋白表达水平。

Figure 3. Effects of different drugs on cell function

下载: 全尺寸图片幻灯片

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