العنوان البديل: Study on the mechanism of transcription factor EB in autophagy of aging cardiomyocytes. (English)

المؤلفون: 盛思琪, 谢琳, 姜怡邓, 熊建团, 杨安宁, 吴凯, 杨勇, 杨晓明

المصدر: Tianjin Medical Journal; Mar2023, Vol. 51 Issue 3, p246-252, 7p

الملخص (بالإنجليزية): Objective To explore the mechanism of transcription factor EB (TFEB) in autophagy of aging cardiomyocytes. Methods Animal experiment: Twenty aged Wistar rats were randomly divided into the sham operation group (Sham group) and the ischemia-reperfusion injury group (I/R group). Cell experiments: (1) Aging cardiomyocytes were cultured in vitro, incubated with 8 g/L D-galactose for 8 days, and then divided into the normoxia group and the hypoxia/ reoxygenation group (H/R group). (2) Aging cardiomyocytes transfected by adenovirus overexpressing and interfering with TFEB were divided into the Ad-GFP group, the Ad-GFP+H/R group, the Ad-TFEB group, the Ad-TFEB+H/R group, the sh-NC group, the sh-NC+H/R group, the sh-TFEB group and the sh-TFEB+H/R group. (3) Aging cardiomyocytes treated with specific inhibitors of DNMT1, DNMT3a and DNMT3b after hypoxia/reoxygenation were divided into the H/R group, the DNMT1 specific inhibitor (DC-05) group, the DNMT3a specific inhibitor (TFD) group and the DNMT3b specific inhibitor (NA) group. (4) Aging cardiomyocytes transfected by adenovirus interfering with DNMT3b were divided into the sh-NC group, the sh-NC+H/R group, the sh-DNMT3b group and the sh-DNMT3b+H/R group. Quantitative real-time PCR (qPCR) was used to detect the mRNA level of TFEB, and Western blot assay was used to detect the autophagy related proteins TFEB, LC3B and p62 in aging cardiomyocytes. The DNA methylation levels of TFEB promoter in aging myocardium and cardiomyocytes were detected by nested methylation specific PCR (nMS-PCR). Results Compared with the Sham group or the normoxia group, the mRNA and protein expression of TFEB were decreased in the I/R group and the H/R group (P＜ 0.01). The protein expression of LC3B-Ⅱ/Ⅰ was decreased in aging cardiomyocytes after overexpression of TFEB in the Ad-TFEB group compared with the Ad-GFP group, while the protein expression of p62 was increased (P＜0.01). The opposite results were obtained after interfering with TFEB (P＜0.01). Compared with the Sham group or the normoxia group, the DNA methylation level of the TFEB promoter was increased in the I/R group and the H/R group (P＜0.05). Compared with the H/R group, it was found that the mRNA and protein expression level of TFEB were increased in the NA group (P＜ 0.01). And the TFEB mRNA and protein expression were increased in aging cardiomyocytes after overexpressed interference with DNMT3b (P＜0.01). Conclusion DNMT3b inhibits the TFEB expression by regulating DNA methylation of TFEB promoter, thus to promote autophagy of aging cardiomyocytes. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 目的 探讨转录因子 EB（TFEB）在衰老心肌细胞自噬中的作用机制。方法 动物实验：将 20 只老年 Wistar大鼠采取随机数字表法分为假手术组（Sham组）和缺血再灌注组（I/R组）。细胞实验：（1）体外培养大鼠心肌细 胞，采用8 g/L D-半乳糖孵育8 d后分为常氧（Normoxia）组和缺氧/复氧（H/R）组。（2）在衰老心肌细胞中分别转染过表 达和干扰TFEB腺病毒分为过表达对照（Ad-GFP）组、过表达对照+缺氧/复氧（Ad-GFP+H/R）组、过表达TFEB（AdTFEB）组、过表达TFEB+缺氧/复氧（Ad-TFEB+H/R）组、干扰对照（sh-NC）组、干扰对照+缺氧/复氧（sh-NC+H/R）组、 干扰TFEB（sh-TFEB）组、干扰TFEB+缺氧/复氧（sh-TFEB+H/R）组。（3）分别用DNMT1、DNMT3a、DNMT3b的特异性 抑制剂DC-05、TFD、NA处理缺氧/复氧后的衰老心肌细胞分为H/R组、DC-05组、TFD组、NA组。（4）在衰老心肌细胞 中转染干扰 DNMT3b 腺病毒分为干扰对照（sh-NC）组、干扰对照缺氧/复氧（sh-NC+H/R）组、干扰 DNMT3b（shDNMT3b）组、干扰DNMT3b缺氧/复氧（sh-DNMT3b+H/R）组。实时荧光定量PCR（qPCR）检测TFEB的mRNA表达水 平；Western blot 检测衰老心肌细胞中自噬相关蛋白TFEB、LC3B 和p62的蛋白表达；巢式甲基化特异性PCR（nMSPCR）检测 TFEB 启动子区的 DNA 甲基化水平。结果 与 Sham 组或 Normoxia 组比较，I/R 组和 H/R 组中 TFEB 的 mRNA和蛋白表达水平降低（P＜0.01）；过表达TFEB后，与Ad-GFP组比较，Ad-TFEB组LC3B-Ⅱ/Ⅰ的蛋白表达水平 降低，p62的蛋白表达水平升高（P＜0.01），而干扰TFEB后得到相反的结果（P＜0.01）。与Sham组或Normoxia组比 较，I/R组和H/R组中TFEB启动子区DNA甲基化水平升高（P＜0.05）。与H/R组比较，NA组TFEB mRNA和蛋白表达 水平升高（P＜0.01）；干扰DNMT3b后，与sh-NC组比较，sh-DNMT3b组TFEB mRNA和蛋白表达水平升高（P＜0.01）。 结论 DNMT3b通过调控TFEB启动子区DNA甲基化抑制TFEB的表达，进而促进衰老心肌细胞自噬。 [ABSTRACT FROM AUTHOR]

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العنوان البديل: The role of FoxO1 in renal podocyte injury and apoptosis induced by homocysteine. (English)

المؤلفون: 张宏红, 谢琳, 盛思琪, 卢冠军, 姜怡邓 1,2,3，, 杨安宁, 李桂忠

المصدر: Tianjin Medical Journal; Jan2022, Vol. 50 Issue 1, p53-58, 6p

الملخص (بالإنجليزية): Objective To discuss the role of forkhead box O1 (FoxO1) in the injury and apoptosis of podocytes induced by homocysteine (Hcy). Methods Ten Cbs^+/+ mice with normal cysteine β -synthase (Cbs) gene and 10 Cbs^+/- mice with single gene knockout were fed a high methionine diet. After 8 weeks, the mice were sacrificed to collect kidney tissues. Morphological changes of glomerulus were observed by PAS staining. Podocytes were cultured in vitro and divided into the control group and the Hcy group (treated with cell culture medium containing 80 μmol/L Hcy for 48 h). Podocytes were transfected with Ad-FoxO1 overexpressed adenovirus and Sh-FoxO1 interfering adenovirus carrying green fluorescent protein (GFP), and they were divided into the control group, the Ad-GFP group, the Ad-FoxO1 group, the Sh-NC group, the Sh-FoxO1 group, the Ad-GFP+Hcy group, the Ad-FoxO1+Hcy group, the Sh-NC+Hcy group and the Sh-FoxO1+Hcy group. Western blot assay was used to detect the expression of Podocin and Nephrin, FoxO1 and apoptosis-related B lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cystine proteinase 12 (Caspase12) proteins in renal tissues and podocytes of mice. The expression of FoxO1 mRNA in mouse kidney tissue and podocytes was detected by qPCR. Results PAS staining results showed that the glomerular structure was normal, the basement membrane was clear and distributed evenly in Cbs^+/+ mice, while the glomerular basement membrane presented intermittent thickening and the mesangial matrix increased in Cbs^+/- mice. Compared with Cbs^+/+ mice, the expression levels of Podocin, Nephrin and FoxO1 protein and FoxO1 mRNA were significantly decreased in Cbs^+/- mice (P＜0.01). Compared with the control group, the expression of FoxO1 protein and mRNA in podocytes were significantly decreased in the Hcy group after treated with Hcy (P＜0.01). After overexpression of FoxO1 in podocytes, compared with the Ad-GFP+Hcy group, the protein expression of Podocin and Nephrin were significantly increased, both the ratio of Bax/Bcl-2 and the protein expression of Caspase12 were significantly decreased in the Ad-FoxO1+Hcy group (P＜0.05). After FoxO1 interference, compared with the Sh-NC+Hcy group, Podocin and Nephrin protein expressions were significantly decreased, Bax/Bcl-2 ratio and Caspase12 protein expression were significantly increased in the Sh-FoxO1+Hcy group (P＜0.05). Conclusion FoxO1 can reduce Hcy- induced renal podocyte injury and apoptosis in mice. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 目的 探讨叉头状转录因子 O1（FoxO1）在同型半胱氨酸（Hcy）诱导的肾脏足细胞损伤及凋亡中的作用。 方法 采用高蛋氨酸饮食喂养胱硫醚 β-合成酶（Cbs）基因正常 Cbs^+/+小鼠和单基因敲除 Cbs+/-小鼠各 10 只。8 周后处 死，收集肾组织，PAS 染色观察小鼠肾小球形态学变化。体外培养足细胞，分为 Control 组和 Hcy 组（用含 80 μmol/L Hcy 的细胞培养液干预 48 h）。将携带绿色荧光蛋白（GFP）的过表达 FoxO1 腺病毒（Ad-FoxO1）和干扰 FoxO1 腺病毒 （Sh-FoxO1）转 染 足 细 胞 ，分 为 对 照 组 、Ad-GFP 组 、Ad-FoxO1 组 、Sh-NC 组 、Sh-FoxO1 组 、Ad-GFP+Hcy 组 、Ad- FoxO1+Hcy 组、Sh-NC+Hcy 组、Sh-FoxO1+Hcy 组。Western blot 检测小鼠肾组织和足细胞裂隙膜蛋白（Podocin 和 Nephrin）、FoxO1 及凋亡相关蛋白 B 淋巴细胞瘤-2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、半胱氨酸蛋白酶 12（Caspase12） 蛋白表达；qPCR 检测小鼠肾组织和足细胞中 FoxO1 mRNA 的表达。结果 PAS 染色结果显示，Cbs^+/+组小鼠肾小球结 构正常，基底膜清晰且分布均匀，而 Cbs^+/-组小鼠肾小球基底膜则呈现节段性增厚，系膜基质增多；与 Cbs^+/+组小鼠比 较，Cbs^+/-组小鼠肾组织中 Podocin、Nephrin 和 FoxO1 蛋白、FoxO1 mRNA 的表达明显降低（P＜0.01）。与 Control 组比 较，Hcy 组 FoxO1 mRNA 和蛋白表达显著降低（P＜0.01）；过表达 FoxO1 后，与 Ad-GFP+Hcy 组相比，Ad-FoxO1+Hcy 组 Podocin 和 Nephrin 蛋白表达均明显升高，Bax/Bcl-2 比值、Caspase12 蛋白表达均明显降低（P＜0.05）；而干扰 FoxO1 后，与 Sh-NC+Hcy 组相比，Sh-FoxO1+Hcy 组 Podocin 和 Nephrin 蛋白表达均明显降低，Bax/Bcl-2 比值、Caspase12 蛋白 表达均明显增高（P＜0.05）。结论 FoxO1 可减轻 Hcy 诱导的小鼠肾脏足细胞损伤及凋亡。 [ABSTRACT FROM AUTHOR]

: Copyright of Tianjin Medical Journal is the property of Tianjin Medical Journal and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)