العنوان البديل: Hypoxic postconditioning protects myocardium by regulating autophagy in aging cardiomyocytes through piRNA-005854. (English)

المؤلفون: 迟宏扬, 杨慧霞, 郝银菊, 杨安宁, 白志刚, 焦 运, 熊建团, 马胜超, 姜怡邓

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 5/8/2024, Vol. 28 Issue 13, p2054-2060, 7p

مصطلحات موضوعية: LACTATE dehydrogenase, ISCHEMIC postconditioning, REPERFUSION injury, MYOCARDIAL injury, WESTERN immunoblotting, GALACTOSE, CREATINE kinase

الملخص (بالإنجليزية): BACKGROUND: Ischemic postconditioning is one of the effective ways to reduce ischemia-reperfusion injury and has been more and more widely used in clinical practice in recent years, but its specific molecular mechanism has yet to be studied. OBJECTIVE: To investigate the role and mechanism of piRNA-005854 in the aging cardiomyocytes caused by hypoxic postconditioning. METHODS: In vitro, cardiomyocytes were administered 8 mg/mL D-galactose for 9 days to induce their aging. β-Galactosidase staining was used to observe the aging of cardiomyocytes. Senescent cells were treated with hypoxia/reoxygenation and hypoxic postconditioning. ELISA was utilized to detect changes in myocardial injury markers creatine kinase isoenzyme MB and lactate dehydrogenase levels. Western blot assay was applied to detect the expression changes of autophagy-related proteins LC3II, p62, ULK1 and phosphorylated ULK1 in aging cardiomyocytes. qRT-PCR was employed to determine the expression level of piRNA-005854. piRNA-005854 inhibitor and piRNA-005854 mimics were transferred into aging cardiomyocytes and followed with hypoxic postconditioning. Western blot assay was used to examine the expression of LC3II, p62, ULK1 and phosphorylated ULK1. RESULTS AND CONCLUSION: (1) D-galactose induced obvious senescence of cardiomyocytes 9 days later. (2) Compared with the normoxia group, creatine kinase isoenzyme MB and lactate dehydrogenase levels increased in the hypoxia/reoxygenation group (P < 0.01); LC3 II/I expression was increased; p62 expression was decreased; ULK1 phosphorylation level was increased, and piRNA-005854 expression was increased (P < 0.01). (3) Compared with the hypoxia/ reoxygenation group, creatine kinase isoenzyme MB and lactate dehydrogenase levels significantly reduced in the hypoxic postconditioning group (P < 0.01); LC3 II/I expression significantly decreased (P < 0.05); p62 expression increased (P < 0.01); ULK1 phosphorylation level decreased (P < 0.05), and piRNA-005854 expression decreased (P < 0.01). (4) After transfection of piRNA-005854 inhibitor, LC3II/I expression was decreased (P < 0.01); the expression of p62 was increased significantly (P < 0.05); the phosphorylation level of ULK1 was decreased significantly (P < 0.01). After transfection of piRNA-005854 mimics, LC3II/ I expression was increased significantly; the expression of p62 was decreased, and the phosphorylation level of ULK1 was increased significantly (P < 0.01). (5) The results show that piRNA-005854-mediated reduction of ULK1-dependent autophagy level is a possible mechanism that hypoxic postconditioning exerts its protective effect on aging cardiomyocytes. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：缺血后处理是减轻缺血再灌注损伤的有效方式之一, 近年来被越来越广泛地应用于临床实践, 但其具体分子机制还有待研究。 目的：探讨piRNA-005854在衰老心肌细胞缺氧后处理中的作用及机制。 方法：体外给予心肌细胞8 mg/mL D-半乳糖9 d诱导其衰老, β-半乳糖苷酶染色观察心肌细胞的衰老情况；衰老后细胞给予缺氧/复氧处 理和缺氧后处理, ELISA检测心肌损伤标志物肌酸激酶同工酶MB以及乳酸脱氢酶水平；Western blot检测衰老心肌细胞中自噬相关蛋白 LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达；qRT-PCR检测piRNA-005854的表达水平；进一步用piRNA-005854 inhibitor及piRNA-005854 mimics 转染衰老心肌细胞并进行缺氧后处理, Western blot检测LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达。 结果与结论：①D-半乳糖诱导9 d后心肌细胞出现明显衰老；②与正常氧组比较, 缺氧/复氧组肌酸激酶同工酶MB以及乳酸脱氢酶水平增 加(P < 0.01)；LC3Ⅱ/Ⅰ表达升高、p62表达降低、ULK1磷酸化水平升高、piRNA-005854表达升高(P < 0.01)；③与缺氧/复氧组比较, 缺氧后 处理组肌酸激酶同工酶MB以及乳酸脱氢酶水平明显减少(P < 0.01)；LC3Ⅱ/Ⅰ表达明显降低(P < 0.05)、p62表达升高(P < 0.01)、ULK1磷酸化 水平降低(P < 0.05)、piRNA-005854表达降低(P < 0.01)；④转染piRNA-005854 inhibitor后, LC3Ⅱ/Ⅰ表达降低(P < 0.01), p62表达明显升高(P < 0.05), ULK1磷酸化水平明显降低(P < 0.01)；转染piRNA-005854 mimics后, LC3Ⅱ/Ⅰ表达显著升高, p62表达降低, ULK1磷酸化水平明显增 加(P < 0.01)；⑤结果表明, piRNA-005854介导的ULK1依赖性自噬水平降低是衰老心肌细胞缺氧后处理发挥保护作用的可能机制。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)

العنوان البديل: Effect of cyclic RNA hsa-circ-0001360 on homocysteine-induced apoptosis of human umbilical vein endothelial cells. (English)

المؤلفون: 况园军, 于素美, 钟颖怡, 章旭红, 马胜超, 杨安宁, 郝银菊, 熊建团, 焦 运, 姜怡邓

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 9/8/2024, Vol. 28 Issue 25, p4060-4064, 5p

الملخص (بالإنجليزية): BACKGROUND: Increased homocysteine level induces apoptosis of human umbilical vein endothelial cells, but the mechanism remains unclear. OBJECTIVE: To investigate the role of hsa-circ-0001360 in human umbilical vein endothelial cell apoptosis induced by homocysteine. METHODS: In vitro cultured human umbilical vein endothelial cells were divided into control group, homocysteine group, interference control group, interference control + homocysteine group, hsa-circ-0001360 interference group, hsa-circ-0001360 + homocysteine interference group, overexpression control group, overexpression control + homocysteine group, hsa-circ-0001360 overexpression group and hsa-circ-0001360 + homocysteine overexpression group. All groups were treated with 100 μmol/L homocysteine. After 72 hours of intervention, the expressions of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blot assay. The apoptotic rate was detected by flow cytometry. Quantitative real-time PCR was used to detect the expression of hsacirc-0001360. RESULTS AND CONCLUSION: (1) Compared with the control group, the expression of Caspase-3 and Bax was significantly increased (P < 0.01), and the expression of Bcl-2 was significantly decreased (P < 0.01), and the apoptotic rate was significantly increased (P < 0.01) in the homocysteine group. (2) Compared with control group, the expression of hsa-circ-0001360 was significantly increased in the homocysteine group (P < 0.01). (3) The expression of hsa-circ-0001360 was significantly higher in the cytoplasm than that in the nucleus (P < 0.01). (4) Compared with the interference control C group and interference control + homocysteine group, the expressions of Caspase-3 and Bax were significantly decreased (P < 0.01), while the expression of Bcl-2 was significantly increased (P < 0.01); the apoptotic rate was significantly decreased (P < 0.01) in sh-hsa-circ-0001360 interference group and sh-hsa-circ-0001360 + homocysteine interference group. (5) Compared with overexpression control group and overexpression control + homocysteine group, the expressions of Caspase-3 and Bax were significantly increased (P < 0.01), while the expression of Bcl-2 was significantly decreased (P < 0.01); the apoptotic rate was significantly increased (P < 0.01) in the hsa-circ-0001360 overexpression group and the hsa-circ-0001360 + homocysteine overexpression group. (6) In conclusion, hsa-circ-0001360 can promote the apoptosis of human umbilical vein endothelial cells induced by homocysteine. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：同型半胱氨酸水平上升会诱导人脐静脉内皮细胞发生凋亡，但机制尚不清楚。 目的：探讨hsa-circ-0001360在同型半胱氨酸诱导人脐静脉内皮细胞发生凋亡中的作用。 方法：体外培养人脐静脉内皮细胞，将其分为对照组、同型半胱氨酸组、干扰对照组、干扰对照+同型半胱氨酸组、干扰hsa-circ-0001360 组、干扰hsa-circ-0001360+同型半胱氨酸组、过表达对照组、过表达对照+同型半胱氨酸组、过表达hsa-circ-0001360组和过表达 hsa-circ-0001360+同型半胱氨酸组，同型半胱氨酸的干预浓度均为100 μmol/L。干预细胞72 h后，应用Western blot检测凋亡相关蛋白Bax、 Bcl-2和Caspase-3的表达；流式细胞仪检测细胞的凋亡率；实时荧光定量PCR(qRT-PCR)检测hsa-circ-0001360的表达水平。 结果与结论：①与对照组相比，同型半胱氨酸组人脐静脉内皮细胞中Caspase-3和Bax的表达明显升高(P < 0.01)，Bcl-2的表达明显降低( P < 0.01)，细胞凋亡率明显增高(P < 0.01)；②与对照组相比，同型半胱氨酸组人脐静脉内皮细胞中hsa-circ-0001360的表达明显升高(P < 0.01)； ③hsa-circ-0001360在人脐静脉内皮细胞的细胞质中表达显著高于细胞核(P < 0.01)；④与干扰对照组或干扰对照+同型半胱氨酸组相比，干 扰hsa-circ-0001360组和干扰hsa-circ-0001360+同型半胱氨酸组人脐静脉内皮细胞中Caspase-3、Bax的表达明显降低(P < 0.01)，Bcl-2的表达明 显升高(P < 0.01)，细胞凋亡率明显降低(P < 0.01)；⑤与过表达对照组或过表达对照+同型半胱氨酸组相比，过表达hsa-circ-0001360组和过 表达hsa-circ-0001360+同型半胱氨酸组人脐静脉内皮细胞中Caspase-3、Bax的表达明显升高(P < 0.01)，Bcl-2的表达明显降低(P < 0.01)，细胞 凋亡率明显升高(P < 0.01)；⑥以上结果表明，hsa-circ-0001360可以促进同型半胱氨酸诱导人脐静脉内皮细胞发生凋亡。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)

العنوان البديل: Involvement of miR-144-3p in Cbs^+/- mouse hepatocyte autophagy induced by high-methionine diet. (English)

المؤلفون: 盛思琪, 谢 琳, 赵翔宇, 姜怡邓, 吴 凯, 熊建团, 杨安宁, 郝银菊, 焦 运

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 3/18/2024, Vol. 28 Issue 8, p1289-1294, 6p

مصطلحات موضوعية: FATTY liver, AUTOIMMUNE hepatitis, PEARSON correlation (Statistics), CELL survival, GENE knockout, ASPARTATE aminotransferase

الملخص (بالإنجليزية): BACKGROUND: High-methionine diet can cause liver injury in Cbs^+/- mice, and hyperhomocystinemia is related to the occurrence and progression of various liver-related diseases, such as hepatic steatosis, autoimmune hepatitis, and alcoholic fatty liver disease. MicroRNAs (miRNAs) are involved in various cellular processes including cell survival, differentiation and autophagy, which are of great significance. OBJECTIVE: To investigate the critical role of miR-144-3p on Cbs^+/- mouse hepatocyte autophagy induced by high methionine die. METHODS: (1) Ten male cystathione-β-synthase normal (Cbs^+/+) mice and another 10 male mice with single gene knockout (Cbs^+/-) of similar body mass, 4 weeks of age, were fed a high-methionine diet and executed after 12 weeks to take liver tissue. (2) Human hepatocytes (HL-7702) were cultured in vitro and divided into control [0 μmol/L homocysteine (Hcy)], Hcy (100 μmol/L Hcy), mimic-NC (transfected with mimic-NC), mimic-NC + Hcy (mimic-NC transfecton+100 μmol/L Hcy), miR-144-3p mimic (transfected with miR-144-3p mimic), and miR-144-3p mimic + Hcy (miR-144-3p mimic transfection+100 μ mol/L Hcy), inhibitor-NC (transfected with inhibitor-NC), inhibitor-NC + Hcy (inhibitor-NC transfection + 100 μmol/L Hcy), miR-144-3p inhibitor (transfected with miR-144-3p inhibitor), and miR-144-3p inhibitor + Hcy (miR-144-3p inhibitor transfection + 100 μmol/L Hcy). Quantitative real-time PCR was used to detect the expression of miR144-3p in liver tissue and hepatocytes. After transfection of miR-144-3p mimic or inhibitor, quantitative real-time PCR and western blot were used to detect the transfection efficiency of miR-144-3p and its effect on the expression of autophagy-related proteins LC3B and p62. The levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were determined by enzyme linked immunosorbent assay. The correlation between the expression of miR-144-3 in hepatocyte and the levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were analyzed by Pearson correlation analysis. RESULTS AND CONCLUSION: Compared with the Cbs^+/+ group and control group, the expression of miR-144-3p in the liver tissue of the Cbs^+/- group and in hepatocytes of the Hcy group was decreased (P < 0.01). The expression of LC3B-II/I was decreased in hepatocyte after transfection of miR-144-3p mimic, while the protein expression of p62 was increased (P < 0.01). The opposite results were obtained after transfection of miR-144-3p inhibitor (P < 0.01). Compared with the mimic-NC group, the levels of alanine transferase and aspartate aminotransferase were decreased in the miR-144-3p mimic group (P < 0.01), while the opposite results were obtained in the inhibitor-NC group (P < 0.01). The expression of miR-144-3p in hepatocytes was negatively correlated with the levels of alanine transferase (P < 0.01, r=-0.887 6) and aspartate aminotransferase (P < 0.01, r=-0.829 9) in the supernatant of hepatocytes. To conclude, Hcy promotes hepatocyte autophagy by inhibiting the expression of miR-144-3p, which subsequently aggravates liver injury. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：高蛋氨酸饮食可导致Cbs^+/- 小鼠发生肝损伤, 高同型半胱氨酸血症与肝脂肪变性、自身免疫性肝炎、酒精性脂肪肝等多种肝脏相关 疾病的发生和进展有关。微小RNA (miRNAs)参与细胞存活、分化和细胞自噬等各种细胞过程, 具有重要意义。 目的：探讨miR-144-3p在高蛋氨酸饮食诱导Cbs^+/- 小鼠肝细胞自噬中的关键作用。 方法：①选取4周龄体质量相近的雄性胱硫醚β-合成酶基因正常(Cbs^+/+)小鼠和单基因敲除(Cbs^+/- )小鼠各10只, 均饲以高蛋氨酸饮食, 12 周后处死, 留取肝脏组织。②体外培养人源肝细胞(HL-7702), 分为对照组(0 μmol/L同型半胱氨酸)、同型半胱氨酸组(100 μmol/L同型半 胱氨酸)、mimic-NC组(转染mimic-NC)、mimic-NC+同型半胱氨酸组(转染mimic-NC+100 μmol/L同型半胱氨酸)、miR-144-3p mimic组(转染 miR-144-3p mimic)、miR-144-3p mimic+同型半胱氨酸组(转染miR-144-3p mimic+100 μmol/L同型半胱氨酸)、inhibitor-NC组(转染inhibitor-NC)、 inhibitor-NC+同型半胱氨酸组(转染inhibitor-NC+100 μmol/L同型半胱氨酸)、miR-144-3p inhibitor组(转染miR-144-3p inhibitor)、miR-144-3p inhibitor+同型半胱氨酸组(转染miR-144-3p inhibitor+100 μmol/L同型半胱氨酸)。采用荧光定量PCR检测肝组织和肝细胞中miR-144-3p的表达 水平；转染miR-144-3p模拟物或抑制剂后, 采用荧光定量PCR和Western blot分别检测miR-144-3p的转染效率及其对LC3B和p62蛋白表达的影 响；酶联免疫法检测肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶的表达情况；Pearson相关性分析肝细胞miR-144-3p表达与肝 细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶含量的相关性。 结果与结论：①与Cbs^+/+组比较, Cbs^+/- 组小鼠肝组织和同型半胱氨酸组肝细胞中miR-144-3p的表达水平降低(P < 0.01)；②转染miR-144-3p 模拟物后, 与mimic-NC比较, miR-144-3p mimic组中LC3B-Ⅱ/Ⅰ蛋白的表达水平降低, p62蛋白的表达水平升高(P < 0.01)；转染miR-144-3p inhibitor后, 得到相反的结果(P < 0.01)；③与mimic-NC组相比, miR-144-3p mimic组肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移 酶的含量降低(P < 0.01)；与inhibitor-NC组比较, 得到相反的结果(P < 0.01)；④肝细胞中miR-144-3p的表达与肝细胞上清液中丙氨酸氨基转 移酶(P < 0.01, r=-0.887 6)和门冬氨酸氨基转移酶(P < 0.01, r=-0.829 9)的含量呈负相关；⑤结果表明, 同型半胱氨酸通过抑制miR-144-3p 的表达促进肝细胞的自噬, 进而加重肝损伤。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)

العنوان البديل: Shikonin inhibits fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p. (English)

المؤلفون: 唐玉婷, 贺 茜, 万 瑀, 王建军, 杨安宁, 吴 凯, 焦 运, 白志刚, 姜怡邓, 沈江涌

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 12/18/2023, Vol. 27 Issue 35, p5642-5648, 7p

مصطلحات موضوعية: HYPERTROPHIC scars, SCARS, DIMETHYL sulfone, GENE expression, CELL migration, CELL culture, COLLAGEN

الملخص (بالإنجليزية): BACKGROUND: Previous studies have shown that shikonin has the potential to treat hypertrophic scar and that microRNAs are involved in the regulation of the pathological mechanism of hypertrophic scar. It is speculated that shikonin may regulate the occurrence and development of hypertrophic scar through microRNAs regulation. OBJECTIVE: To investigate the mechanism of shikonin on the fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p. METHODS: Hypertrophic scar tissue and normal skin tissue adjacent to the scar (within 3 cm around the scar) were provided by the Department of Burn and Plastic Surgery, General Hospital of Ningxia Medical University to extract human hypertrophic scar fibroblasts and human normal skin fibroblasts, respectively. Hematoxylin-eosin staining was used to identify normal skin and hypertrophic scar and immunofluorescence was used to identify fibroblasts. The relative expression level of MicroRNA-382-5p was detected by quantitative real-time PCR at the tissue level. Hypertrophic scar fibroblasts were randomly divided into shikonin group (shikonin was dissolved in dimethyl sulfone to make drug solution at a concentration of 13.46 μmol/L, which was used for cell culture for 24 hours), dimethyl sulfone group, MicroRNA-382-5p negative control group, MicroRNA-382-5p inhibitor group, shikonin+MicroRNA-382-5p negative control group and shikonin+MicroRNA-382-5p overexpression group. Real-time fluorescence quantitative PCR and western blot were used to detect the expression of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin at mRNA and protein levels, respectively. Cell counting kit-8 was used to detect cell viability. Cell scratch test was used to detect the migration ability of cells. RESULTS AND CONCLUSION: Compared with normal skin fibroblasts, hypertrophic scar fibroblasts had stronger proliferative activity (P < 0.01). Compared with the dimethyl sulfone group, shikonin down-regulated the expression levels of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in human hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and inhibited the migration of hypertrophic scar fibroblasts (P < 0.05). Compared with normal skin, MicroRNA-382-5p was highly expressed in hypertrophic scar (P < 0.01), and shikonin could down-regulate the expression of MicroRNA-382-5p (P < 0.01). Inhibition of MicroRNA-382-5p down-regulated the expression level of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and inhibited the migration of hypertrophic scar fibroblasts (P < 0.05). Under the influence of shikonin, overexpression of MicroRNA-382-5p upregulated the expression levels of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and promoted the migration of hypertrophic scar fibroblasts (P < 0.01). To conclude, shikonin can inhibit the fibrosis and migration of hypertrophic scar fibroblasts by down-regulating the expression of MicroRNA-382-5p. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：目前已有研究表明紫草素具有治疗增生性瘢痕的潜力,且miRNA参与增生性瘢痕的病理机制调控,猜测紫草素有可能通过影响 miRNA调控增生性瘢痕的发生发展. 目的：探讨紫草素通过影响MicroRNA-382-5p对人增生性瘢痕成纤维细胞纤维化的作用机制. 方法：收集宁夏医科大学总医院烧伤整形外科提供的增生性瘢痕组织及瘢痕旁正常皮肤组织(瘢痕旁3 cm以内),并分别分离出人增生性瘢 痕成纤维细胞和正常成纤维细胞用于后续实验.苏木精-伊红染色鉴定正常皮肤和增生性瘢痕；免疫荧光鉴定成纤维细胞；采用实时荧光 定量PCR从组织水平检测MicroRNA-382-5p相对表达水平.将增生性瘢痕成纤维细胞随机分为紫草素组(紫草素溶于二甲基亚砜配成浓度为 13.46 μmol/L的药物干预细胞24 h),二甲基亚砜组,MicroRNA-382-5p阴性对照组,MicroRNA-382-5p抑制组,紫草素+MicroRNA-382-5p阴性 对照组及紫草素+MicroRNA-382-5p过表达组.实时荧光定量PCR检测Ⅰ型胶原蛋白α1,Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白mRNA表达； Western blot检测Ⅰ型胶原蛋白α1,Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的蛋白表达；CCK-8法检测细胞活性；划痕实验检测细胞迁移能力. 结果与结论：①与正常皮肤成纤维细胞相比,增生性瘢痕成纤维细胞增殖活性更强(P < 0.01)；②与二甲基亚砜组相比,紫草素组可以下调增 生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1,Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA：P < 0.01,蛋白：P < 0.01),并抑制增生性 瘢痕成纤维细胞迁移(P < 0.05)；③与正常皮肤相比,MicroRNA-382-5p在增生性瘢痕中呈高表达(P < 0.01),且紫草素能下调增生性瘢痕成纤维 细胞中MicroRNA-382-5p的表达(P < 0.01)；④敲低MicroRNA-382-5p能下调增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1,Ⅲ型胶原蛋白α1和α-平滑 肌肌动蛋白的表达水平(mRNA：P < 0.01,蛋白：P < 0.01),并抑制增生性瘢痕成纤维细胞迁移(P < 0.05)；⑤过表达MicroRNA-382-5p能上调在紫 草素影响下增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1,Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA：P < 0.01,蛋白：P < 0.01),并促 进增生性瘢痕成纤维细胞迁移(P < 0.01)；⑥提示紫草素可通过下调MicroRNA-382-5p的表达抑制增生性瘢痕成纤维细胞的纤维化和迁移. [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)

العنوان البديل: Erastin inhibits proliferation of hypertrophic scar fibroblasts. (English)

المؤلفون: 贺 茜, 万 瑀, 唐玉婷, 杨安宁, 吴 凯, 焦 运, 白志刚, 姜怡邓, 沈江涌

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 11/18/2023, Vol. 27 Issue 32, p5120-5125, 6p

مصطلحات موضوعية: PROLIFERATING cell nuclear antigen, HYPERTROPHIC scars, CYCLIN-dependent kinase inhibitors, FERRITIN, GENE expression, IRON ions

الملخص (بالإنجليزية): BACKGROUND: Hypertrophic scar is a kind of pathological scar that appears in the healing process after skin trauma caused by various reasons and there is no effective treatment. OBJECTIVE: To investigate the effect of ferroptosis inducer (Erastin) on the proliferation of human hypertrophic scar fibroblasts. METHODS: Hypertrophic scar samples provided by the Burn Plastic Surgery Department of the General Hospital of Ningxia Medical University and normal skin samples of the same individual were collected. Human hypertrophic scar fibroblasts were then extracted for subsequent experiments. (1) The cells were divided into control group (without treatment) and ferroptosis inducer group (treated with 20 μmol/L Erastin for 24 hours). The expression of Ferritin was detected by western blot. Iron ion detection kit was used to measure cellular iron ion concentration. Malondialdehyde detection kit was used to detect cellular malondialdehyde content. (2) The cells were divided into control group (without treatment), ferroptosis inducer group (treated with 20 μmol/L Erastin for 24 hours) and ferroptosis inducer+ferroptosis inhibitor group (co-treated with 20 μmol/L Erastin and 20 μmol/L Ferrostatin-1 for 24 hours). The mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor (p27) were detected using qRT-PCR and western blot. Cell counting kit-8 and EdU were used to detect cell proliferation viability and levels. RESULTS AND CONCLUSION: Compared with the control group, Erastin decreased the ferritin expression (P < 0.01), increased the content of iron ions (P < 0.05), and elevated the malondialdehyde content in the cells (P < 0.01). Compared with the control group, Erastin decreased the expression of PCNA (P < 0.01), increased the expression of p27 (P < 0.05), weakened the cell proliferation ability (P < 0.01), and reduced the number of EdU-positive cells (P < 0.01). Compared with the ferroptosis inducer group, the ferroptosis inducer + ferroptosis inhibitor group had increased expression of PCNA (P < 0.05), decreased p27 expression (P < 0.05), enhanced cell proliferation (P < 0.01), and increased number of EdU-positive cells (P < 0.05). To conclude, the ferroptosis inducer (Erastin) induces ferroptosis in hypertrophic scar fibroblasts and then inhibits their proliferation. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：增生性瘢痕是各种原因导致的皮肤创伤后愈合过程中出现的一种病理性瘢痕，目前缺乏特效治疗方法。 目的：探讨铁死亡诱导剂Erastin对人增生性瘢痕成纤维细胞增殖的影响。 方法：收集宁夏医科大学总医院烧伤整形外科提供的增生性瘢痕组织和同一个体正常皮肤，提取人增生性瘢痕成纤维细胞进行后续实验。 将细胞分为对照组(不做处理)和铁死亡诱导剂组(用20 μmol/L的Erastin干预细胞24 h)，Western blot检测铁蛋白(Ferritin)的表达，铁离子检 测试剂盒测定细胞铁离子浓度，丙二醛检测试剂盒检测细胞丙二醛水平；将细胞分为对照组(不做处理)、铁死亡诱导剂组(用20 μmol/L的 Erastin干预细胞24 h)和铁死亡诱导剂+铁死亡抑制剂组(用20 μmol/L的Erastin和20 μmol/L的Ferrostatin-1同时干预细胞24 h)，qRT-PCR检测 PCNA及p27的mRNA表达，Western blot检测PCNA及p27的蛋白表达，CCK-8、EdU检测细胞增殖活力和增殖水平。 结果与结论：①与对照组相比，铁死亡诱导剂组铁蛋白减少(P < 0.01)，铁离子增多(P < 0.05)，丙二醛增多(P < 0.01)；②与对照组相比，铁 死亡诱导剂组PCNA的表达降低(P < 0.01)，p27的表达增加(P < 0.05)，细胞增殖能力减弱(P < 0.01)，EdU阳性细胞数减少(P < 0.01)；③与铁死 亡诱导剂组相比，铁死亡诱导剂+铁死亡抑制剂组PCNA的表达增加(P < 0.05)，p27的表达降低(P < 0.05)，细胞增殖能力增强(P < 0.01)，EdU 阳性细胞数增多(P < 0.05)；④结果表明，铁死亡诱导剂Erastin通过诱导增生性瘢痕成纤维细胞发生铁死亡进而抑制其增殖。 [ABSTRACT FROM AUTHOR]

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العنوان البديل: Ferroptosis participates in renal damage induced by high methionine diet in ApoE^-/- mice. (English)

المؤلفون: 畅思容, 孙岳巴, 杨安宁, 李媛媛, 巴王梦, 宝瑞, 余梦雪, 刘志宏

المصدر: Modern Preventive Medicine; 2023, Issue 17, p3133-3150, 7p

مصطلحات موضوعية: GLUTAMATE transporters, APOLIPOPROTEIN E, P53 protein, RENAL fibrosis, GENE expression

الملخص (بالإنجليزية): Objective To investigate whether ferroptosis is involved in renal damage in Apo E~f~ mice induced by high methionine diet. Methods C57BL/6J mice fed with normal diet were used as normal control group, ApoE, mice were randomly divided into model control group (fed with normal diet) and high methionine group (fed with high methionine diet), with 6 mice in each group. Renal injury and fibrosis in mice were observed by PAS and Masson staining. Detection of Fibronectin content by immunofluorescence staining and iron content was measured by colorimetry in renal tissue. The content of malondialdehyde (MDA) in renal tissue was detected by immunofluorescence staining and Thiobarbituric acid (TBA) method. Protein and mRNA expression of p53, SLC7A11, and GPX4 were determined by Western blotting and qRT-PCR. One -way ANOVA was used to compare multiple groups, and Tukey test was used for multiple comparisons. Results The staining results showed that apolipoprotein E (ApoE) gene deletion caused renal extracellular matrix deposition and renal injury, while high methionine diet further aggravated renal extracellular matrix deposition and renal injury. Compared with the normal control group, the content of Fibronectin in the model control group and high methionine group increased (於87.330, P<0.001), the content of iron ion (F=l 11.200, P<0.001) and MDA (7=205.200, P<0»001) increased, the protein level of p53 increased 0=32.380; P<0,001), the level of mRNA increased (F=24.840, P<0.001), the protein level of SLC7A11 and GPX4 decreased (slc7all=18.620, P<0,001; 74=20.830, P<0.001), and mRNA level decreased (码皿口=11200; P<0,001; F(赵4=23.530, P< 0.001). Compared with the model control groups the above indexes changed more significantly in the high methionine group (P<0.05). Conclusion Ferroptosis is involved in renal damage in Apo E~l~ mice induced by high methionine diet. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 目的 探讨铁死亡是否参与高蛋氨酸饮食所致的心。旷小鼠肾脏损伤。方法C57BU6J普通饲料喂养小鼠作为 正常对照组、将心。旷小鼠随机分为模型对照组(普通饲料喂养)和高蛋氨酸组筒蛋氨酸饲料喂养), 每组6只小鼠。PAS 和Masson染色观察小鼠肾脏损伤以及纤维化情况;免疫荧光染色检测肾组织纤维连接蛋白(fibronectin)含量;比色法检 测肾组织二价铁离子含量;免疫荧光染色和硫代巴比妥酸(tliiobarbituric acid, TBA)法检测肾组织丙二醛(malondialdehyde, MDA)#*;Western blotting和qRT-PCR检测p53、SLC7All和GPX4的蛋白以及mRNA表达水平,多组数据比较 采用单因素方差分析, 多重比较采用Tukey检验。结果 染色结果显示, 载脂蛋白E(apolipoprotein E, ApoE)因缺失引 起肾脏细胞外基质沉积, 出现肾损伤, 而高蛋氨酸饮食则进一步加重了肾脏细胞外基质沉积,肾损伤加重;与正常对照 组相比, 模型对照组和高蛋氨酸组Fibronectin含量增加(&87.330, Pv0.001),铁离子(F= 111.200,0.001)和MDA (f =205.200,P<0.001)含量增加, p53 的蛋白水平增加(F=32.380, Pv0.001)、mRNA 水平增加(F=24.840, Pv0.001), SLC7A11 和 GPX4 的蛋白水平降低(殛如=18.620, PvO.001;&齡=20.830, Pv0.001)、mRNA 水平降低(殛如=11.200, Pv 0.001; Fc妒23.530, PvO.001);与模型对照组相比, 以上指标在高蛋氨酸组变化更显著(Pv0.05)。结论铁死亡参与了 高蛋氨酸饮食所致的小鼠肾脏损伤. [ABSTRACT FROM AUTHOR]

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العنوان البديل: Ferroptosis is involved in the pathogenesis of liver injury induced by high methionine diet in ApoE^-/- mice. (English)

المؤلفون: 李媛媛, 孙 岳, 宝 瑞, 畅思容, 王 梦, 余梦雪, 杨安宁, 刘志宏

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 6/18/2023, Vol. 27 Issue 17, p2681-2686, 6p

مصطلحات موضوعية: ALANINE aminotransferase, LIVER cells, LIVER proteins, IRON ions, LIVER injuries, ASPARTATE aminotransferase, GLUTATHIONE peroxidase

الملخص (بالإنجليزية): BACKGROUND: Homocysteine can promote the occurrence of liver injury through oxidative stress, inflammatory response, and endoplasmic reticulum stress. High methionine diet-induced apolipoprotein gene knockout (ApoE^-/-) can induce liver injury and increase in vivo homocysteine level in mice. Ferroptosis is a cellular regulatory death pathway that depends on intracellular iron and causes excessive accumulation of lipid peroxides. However, whether it is involved in the formation of liver injury induced by high methionine diet needs to be further studied and discussed. OBJECTIVE: To explore the role of ferroptosis in hyperhomocysteine-induced liver injury in ApoE^-/- mice. METHODS: Twelve ApoE^-/- mice aged 6 to 8 weeks were randomly divided into control group and high methionine group (n=6 per group), and fed with normal diet and high methionine diet for 13.5 weeks. Liver injury in ApoE^-/- mice was evaluated by pathological observation and detection of aspartate aminotransferase and alanine aminotransferase activity in liver tissue. Iron ion concentration in liver tissue of mice was tested by tissue iron detection kit. Malondialdehyde content and fluorescence intensity were detected to evaluate the degree of lipid peroxidation in the liver tissue of mice. Real-time fluorescence quantitative PCR and western blot were performed to determine the expression of P53 and glutathione peroxidase 4 at mRNA and protein levels in the liver tissue. RESULTS AND CONCLUSION: Compared with the control group, a large number of liver cells in the high methionine diet group were found to have the typical histopathological changes of liver injury, such as disordered arrangement of liver cells, enlarged space, and loose cytoplasm. Compared with the control group, the activities of aspartate aminotransferase and alanine aminotransferase were significantly increased (P < 0.01). Meanwhile, the concentration of iron ions (P < 0.01), malondialdehyde content (P < 0.01), and fluorescence intensity in liver tissue were also significantly increased in the high methionine diet group. Real-time fluorescence quantitative PCR and western blot results showed that the expression level of glutathione peroxidase 4 was decreased (P < 0.01) and the expression of P53 was increased (P < 0.05) in ApoE^-/- mice in the high methionine diet group. These findings indicate that ferroptosis is involved in the pathogenesis of liver injury in ApoE^-/- mice induced by high methionine diet. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：高同型半胱氨酸可以通过氧化应激、炎症反应和内质网应激等机制促进肝损伤的发生。高蛋氨酸饮食诱导的E型载脂蛋白基因敲除 (ApoE^-/-)小鼠可以引起肝损伤以及体内同型半胱氨酸水平升高，铁死亡是一种依赖于细胞内的铁并引起脂质过氧化物过量蓄积的细胞调节 性死亡途径。然而，它是否参与了高蛋氨酸饮食诱导肝损害的形成需进一步研究和探讨。 目的：探讨铁死亡在 ApoE^-/-小鼠高同型半胱氨酸致肝损伤中的作用。 方法：12 只 6-8 周龄雄性 ApoE^-/-小鼠，体质量20-25 g，随机分为对照组和高蛋氨酸组，每组6只，分别采用普通饲料和高蛋氨酸饲料喂养 13.5周。喂养结束后通过病理学观察以及肝组织中天冬氨酸转氨酶、丙氨酸转氨酶活性检测评估小鼠肝损伤情况；组织铁检测试剂盒测 定小鼠肝组织中的铁离子含量；通过肝组织中丙二醛含量和荧光强度评估小鼠肝组织中脂质过氧化程度；实时荧光定量 PCR与Western blotting 方法分别检测两组小鼠肝组织中P53和谷胱甘肽过氧化物酶4的mRNA和蛋白表达水平。 结果与结论：①与对照组相比，高蛋氨酸组发现大量肝细胞排列紊乱，间隙增大，胞浆疏松的典型肝损伤组织病理学改变；天冬氨酸转 氨酶以及丙氨酸转氨酶活性升高(P < 0.01)；肝组织中铁离子含量增加(P < 0.01)，丙二醛含量(P < 0.01)及荧光强度均增加；②实时荧光定量 PCR与Western blotting检测发现，高蛋氨酸组小鼠肝组织中谷胱甘肽过氧化物酶4的表达水平降低(P < 0.01)，P53的表达水平升高(P < 0.05)； ③提示铁死亡参与高蛋氨酸饮食诱导ApoE^-/-小鼠肝损伤的发病过程。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)

العنوان البديل: Shikonin inhibits collagen deposition of human hypertrophic scar fibroblasts via PTEN. (English)

المؤلفون: 唐玉婷, 马芳, 贺茜, 王建军, 杨安宁, 吴凯, 焦运, 白志刚, 姜怡邓, 沈江涌

المصدر: Journal of Practical Medicine / Shiyong Yixue Zazhi; 6/10/2023, Vol. 39 Issue 11, p1382-1388, 7p

الملخص (بالإنجليزية): Objective To investigate the effect of shikonin on the collagen deposition of human hypertrophic scar fibroblasts from the perspective of PTEN. Methods Hematoxylin-eosin staining and Masson staining were used to identify hypertrophic scar tissue. Immunofluorescence was used to identify fibroblasts. The relative expression level of PTEN was detected by Western blot at the tissue level. Hypertrophic scar fibroblasts were randomly divided into dimethyl sulfone group, shikonin group, control group, si-NC group, shikonin+ si-NC group and shikonin+si-PTENC group, western blot were used to detect the expression of PTEN and collagen-related proteins (Col1, Col3 and α-SMA）at protein levels, respectively. Results Compared with normal skin, PTEN was lower expressed in hypertrophic scar (P < 0.05). shikonin was dissolved in dimethyl sulfone to make drug solution at IC50 concentration (13.46 μmol/L), which was used for human hypertrophic scar fibroblasts cell culture for 24 hours, shikonin down-regulated the expression levels of Col1, Col3 and α-SMA in human hypertrophic scar fibroblasts, while up-regulated the expression of PTEN (P < 0.05). Transfected with si-PTEN, the expressions of Col1, Col3 and α-SMA in human hypertrophic scar fibroblasts were increased compared with the si-NC group (P < 0.05). After co-transfection with shikonin + si-PTEN, the expressions of Col1, Col3 and α-SMA in human hypertrophic scar fibroblasts were increased compared with shikonin + si-NC group (P < 0.05). Conclusion Shikonin can inhibit collagen deposition in hypertrophic scar fibroblasts by up-regulating PTEN expression. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 目的 从 PTEN 角度探讨紫草素对人增生性瘢痕成纤维细胞胶原沉积的影响。方法 苏木 精-伊红染色 (HE 染色) 和马松染色 (Masson 染色) 鉴定增生性瘢痕组织, 免疫荧光鉴定人增生性瘢痕成纤 维细胞 (HSFBs); Western blot 检测人增生性瘢痕 (HS) 和同一个体正常皮肤 (NS) 组织中 PTEN 表达水平; 将 HSFBs 随机分组为：DMSO 组、shikonin 组、control 组、si-NC 组、si-PTEN 组、shikonin+si-NC 组 和 shikonin+ si-PTEN 组; Western blot 检测各组中 PTEN 和胶原相关蛋白 (Col1、Col3、α⁃SMA) 的表达。结果 与正 常皮肤组织相比, PTEN 在增生性瘢痕组织中呈低表达 (P < 0.05); 用 IC50 浓度 (13.46 μmol/L) 的紫草素干 预人增生性瘢痕成纤维细胞 24 h 后, 人增生性瘢痕成纤维细胞中 Col1、Col3 和 α-SMA 的表达降低 (P < 0.05), 而 PTEN 的表达升高 (P < 0.05); 转染 si-PTEN 后, 与 si-NC 组相比, 人增生性瘢痕成纤维细胞中 Col1, Col3 和 α⁃SMA 的表达升高 (P < 0.05); 共转染 shikonin + si-PTEN 后, 与 shikonin +si-NC 组相比, 人增生性瘢 痕成纤维细胞中 Col1、ol3 和 α⁃SMA 的表达升高 (P < 0.05)。结论 紫草素可通过上调 PTEN 的表达抑制 增生性瘢痕成纤维细胞的胶原沉积。 [ABSTRACT FROM AUTHOR]

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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: Construction, phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice. (English)

المؤلفون: 郝 玮, 孙 岳, 杨安宁, 刘太阳, 宝 瑞, 刘耀阳, 王秋实, 王 梦, 畅思容, 李媛媛, 刘志宏

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 7/18/2022, Vol. 26 Issue 20, p3207-3211, 5p

الملخص (بالإنجليزية): BACKGROUND: PDHA1 gene is an indispensable gene in the aerobic respiratory chain. The energy metabolism of vascular endothelial cells is related to many diseases. Construction of vascular endothelial cell-specific PDHA1 knockout mice helps to study the role of energy metabolism in diseases related to vascular endothelial cells. OBJECTIVE: To breed vascular endothelial cell-specific PDHA1 knockout mice and identify their phenotypes. METHODS: PDHA1^{(iΔEC/ iΔEC)} mice were co-bred with PDHA1^(iΔEC/-) mice, and more PDHA1^{(iΔEC/ iΔEC)} mice were obtained for subsequent experiments. PCR and agarose gel electrophoresis were used for gene identification. RT-PCR and western blot were used to detect the mRNA and protein expression of genes related to energy metabolism after PDHA1 knockout, respectively. The protein expression of PDHA1 after PDHA1 conditional knockout was determined by double immunofluorescence staining of VE-cadherin and PDHA1. RESULTS AND CONCLUSION: Genotypes of offspring mice were successfully detected by agarose gel electrophoresis, including 15 PDHA1^{(iΔEC/ iΔEC)} and 2 PDHA1^(iΔEC/-) mice. Results of reverse transcription PCR and western blot assay showed that the transcription and translation levels of PDHA1 were decreased, the protein and mRNA expressions of HK2 in the glycolysis pathway were increased, and the protein and mRNA expression of IDH in the aerobic phosphorylation pathway were decreased. Double immunofluorescence staining of VE-cadherin and PDHA1 showed a decrease in the expression of PDHA1. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景: PDHA1基因是有氧呼吸链中不可或缺的基因, 血管内皮细胞的能量代谢与很多疾病有关。构建血管内皮细胞PDHA1基因敲除鼠, 有 助于研究能量代谢在血管内皮细胞相关疾病中的作用。 目的: 繁育血管内皮细胞PDHA1基因特异性敲除小鼠并进行表型鉴定。 方法: 将PDHA1^{(iΔEC/iΔEC)}小鼠与PDHA1^{(iΔEC /-)} 小鼠进行合笼繁育, 获得更多的PDHA1^{(iΔEC/iΔEC)}小鼠进行后续实验。利用PCR和琼脂糖凝胶电泳进行 基因鉴定, 然后利用RT-PCR和Western blot检测敲除PDHA1后与能量代谢相关基因的mRNA及蛋白表达, 利用VE-Cadherin与PDHA1双重免疫 荧光判断PDHA1基因条件性敲除后PDHA1蛋白表达变化。 结果与结论: 利用琼脂糖凝胶电泳实验成功检测出子代小鼠基因型, 包括 PDHA1^{(iΔEC/iΔEC)}15只和PDHA1^(iΔEC/-) 2只小鼠；RT-PCR和Western Blot 检测发现PDHA1在转录水平和翻译水平都发生了下降, 参与糖酵解途径的HK2蛋白及mRNA表达上升, 参与有氧磷酸化途径方向IDH蛋白及 mRNA表达下降；VE-cadherin和PDHA1双重免疫荧光共染色检测到PDHA1的表达下降。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research 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.)