青光安含药血清对转化生长因子-β1诱导人眼Tenon 囊成纤维细胞自噬相关基因表达的影响
Effects of Qingguang'an containing serum on the expression levels of autophagy-related genes in human Tenon's fibroblasts induced by transforming growth factor beta 1.
机构信息
Ophthalmology Department, the First Affiliated Hospital, Hunan University of TCM, Changsha 410007, China.
Eye Research Centre, Hunan University of Traditional Chinese Medicine, Changsha 410208, China.
出版信息
J Tradit Chin Med. 2021 Apr;41(2):236-245.
OBJECTIVE
To explore the effects of Qingguang'an () containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1 (TGF-β1)-activated human Tenon's fibroblasts (HTFs).
METHODS
(a) Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry, which established a cell model after Glaucoma filtration surgery (GFS). (b) The cell models were divided into 4 group: normal group (normal cells), model group (+TGF-β1),treatment group (+TGF-β1+ medicated serum), and positive control group (TGF-β1+ rapamycin). Then, Qingguang'an medicated serum with optimum concentration was added to the corresponding group. The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging. And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry. The expression levels of autophagy related genes － Beclin-1, autophagy related gene 5 (ATG-5), and microtubule-associated protein 1 light chain 3 (LC-3Ⅱ were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.
RESULTS
Compared with the normal group and the model group, the relative mRNA expression levels of autophagy-related genes (Beclin-1, ATG-5 and LC-3Ⅱ in the experimental group were notably increased (P < 0.05, P < 0.01), and with the extension of treatment time, it had an increasing trend (48 h was more obvious), which showed a certain time dependency; the protein expression levels of autophagy-related genes (Beclin-1, ATG-5, and LC-3Ⅱ were significantly increased in the experimental group (P < 0.05, P < 0.01). With the prolongation of treatment time, there was an increasing trend (48 h was relatively obvious), and it revealed a certain time dependency.
CONCLUSION
The Qingguang'an medicated serum could up-regulate autophagy related genes (Beclin1, ATG5, and LC3Ⅱ in the TGF-β1-activated HTFs.
目的
探讨青光安含药血清对转化生长因子β1（TGF-β1）激活的人眼Tenon 氏成纤维细胞（HTFs）中自噬相关基因表达水平的影响。
方法
（a）通过 TGF-β1 刺激原代 HTFs 并进行免疫组织化学染色，建立青光眼滤过术后（GFS）细胞模型。（b）将细胞模型分为 4 组：正常组（正常细胞）、模型组（+TGF-β1）、治疗组（+TGF-β1+含药血清）和阳性对照组（+TGF-β1+雷帕霉素）。然后，向相应组中加入最佳浓度的青光安含药血清。荧光显微镜下用 Cyto-ID 自噬检测试剂盒鉴定自噬阳性细胞，并用 Cytation 5 多功能细胞成像仪进行细胞成像。通过流式细胞术测定自噬阳性细胞的平均荧光强度。采用定量逆转录-聚合酶链反应和 Western blot 分析检测自噬相关基因-Beclin-1、自噬相关基因 5（ATG-5）和微管相关蛋白 1 轻链 3（LC-3Ⅱ）的表达水平。
结果
与正常组和模型组相比，实验组自噬相关基因（Beclin-1、ATG-5 和 LC-3Ⅱ）的相对 mRNA 表达水平明显升高（P<0.05，P<0.01），且随着治疗时间的延长呈递增趋势（48 h 时更为明显），具有一定的时间依赖性；实验组自噬相关基因（Beclin-1、ATG-5 和 LC-3Ⅱ）的蛋白表达水平明显升高（P<0.05，P<0.01）。随着治疗时间的延长，呈递增趋势（48 h 时相对明显），具有一定的时间依赖性。
结论
青光安含药血清可上调 TGF-β1 激活的 HTFs 中自噬相关基因（Beclin1、ATG5 和 LC3Ⅱ）的表达。
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