3-甲基腺嘌呤通过抑制AKT信号减轻糖尿病小鼠的早期肾损伤

doi:10.12122/j.issn.1673-4254.2024.07.03

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (7): 1236-1242.doi: 10.12122/j.issn.1673-4254.2024.07.03

3-甲基腺嘌呤通过抑制AKT信号减轻糖尿病小鼠的早期肾损伤

刘本菊¹(), 王业磊¹^,³(), 任海文²^,⁴, 欧丽雯¹, 邓轩¹, 黄梦欣¹, 吴鑫¹, 龚权²()

^1.长江大学医学部，人体解剖学教研室，湖北荆州 434023
^2.长江大学医学部，免疫学教研室，湖北荆州 434023
^3.重庆市彭水自治县人民医院内科，重庆彭水 409600
^4.重庆医科大学附属璧山医院检验科，重庆璧山 402760

收稿日期:2024-01-07 出版日期:2024-07-20 发布日期:2024-07-25
通讯作者: 龚权 E-mail:liubenju1981@aliyun.com;568862187@qq.com;gongquan1998@163.com
作者简介:刘本菊，博士，讲师，E-mail: liubenju1981@aliyun.com
王业磊，在读硕士研究生，副主任医师，E-mail: 568862187@qq.com
第一联系人：刘本菊、王业磊共同为第一作者
基金资助:
国家自然科学基金(82270893);长江大学2022年大学生创新训练项目(Yz2022300)

3-Methyladenine alleviates early renal injury in diabetic mice by inhibiting AKT signaling

Benju LIU¹(), Yelei WANG¹^,³(), Haiwen REN²^,⁴, Liwen OU¹, Xuan DENG¹, Mengxin HUANG¹, Xin WU¹, Quan GONG²()

^1.Department of Human Anatomy,
^2.Department of Immunology, Medical College of Yangtze University, Jingzhou 434023, China
^3.Department of Internal Medicine, Chongqing Pengshui Autonomous County People's Hospital, Pengshui 409600, China,
^4.Clinical Laboratory, Affiliated Bishan Hospital of Chongqing Medical University, Bishan 402760, China

Received:2024-01-07 Online:2024-07-20 Published:2024-07-25
Contact: Quan GONG E-mail:liubenju1981@aliyun.com;568862187@qq.com;gongquan1998@163.com
Supported by:
National Natural Science Foundation of China(82270893)

摘要/Abstract

摘要：

目的探究3-甲基腺嘌呤（3-MA）减轻糖尿病早期肾损伤的机制。方法将24只小鼠随机分为对照组（CON组，n=8）和模型组（n=16）。模型组采用腹腔注射链脲佐菌素（STZ）诱导糖尿病，CON组注射等体积柠檬酸缓冲液。将造模成功的小鼠随机分为糖尿病组（DM组，n=8）和3-MA干预糖尿病组（DM+3-MA组，n=8），分别灌胃给予等体积生理盐水和3-MA 10 mg/（kg·d），每周记录小鼠体质量和空腹血糖，干预6周后取肾，记录肾/体质量比，PAS染色观察肾小球大小，Western blotting和免疫组化法检测肾皮质α-平滑肌肌动蛋白（α-SMA）和增殖细胞核抗原（PCNA）蛋白表达。将SV40 MES 13细胞分别采用5.6 mmol/L正常糖（NG组）、5.6 mmol/L正常糖+24.4 mmol/L甘露醇（NG+M组）、30 mmol/L高糖（HG组）和30 mmol/L高糖加5 mmol/L 3-MA（HG+3-MA组）培养24 h，CCK8法检测细胞活力，Western blotting检测PCNA蛋白表达。对糖尿病肾脏疾病（DKD）基因靶点与3-MA预测基因靶点的交集基因进行生信分析，并外采用Western blotting对生信分析结果进行验证。结果体内实验结果显示，3-MA具有短期降血糖作用，可减轻糖尿病小鼠肾/体质量比和肾小球肥大（均P<0.05），减少糖尿病小鼠肾皮质α-SMA和PCNA蛋白表达（P<0.01，P<0.001）。体外实验结果显示，3-MA可显著抑制高糖处理的SV40 MES 13细胞活力和PCNA表达（均P<0.001）。生信分析结果显示，蛋白激酶B1（AKT1）为3-MA作用于DKD的关键基因，Western blotting结果显示，3-MA在体内外均可抑制AKT及核糖体蛋白S6（S6）的磷酸化。结论 3-MA通过抑制AKT信号减少系膜细胞增殖减轻糖尿病早期肾损伤。

关键词: 3-甲基腺嘌呤, 糖尿病肾脏疾病, 系膜细胞, 肾损伤

Abstract:

Objective To explore the mechanism of 3-methyladenine (3-MA) for alleviating early diabetic renal injury. Methods Mouse models of streptozotocin (STZ)-induced diabetes mellitus were randomized into model group and 3-MA treatment group for daily treatments with normal saline and 10 mg/kg 3-MA by gavage for 6 weeks, respectively. Body weight and fasting blood glucose of the mice were recorded every week. After the treatments, the kidneys of the mice were collected for measurement kidney/body weight ratio, examination of glomerular size with PAS staining, and detection of α-SMA and PCNA expressions using Western blotting and immunohistochemistry. SV40 MES 13 cells cultured in normal glucose (5.6 mmol/L) and high glucose (30 mmol/L) were treated with 24.4 mmol/L mannitol and 5 mmol/L 3-MA for 24 h, respectively, and the changes in cell viability and PCNA expression were examined using CCK8 assay and Western blotting. Bioinformatics analysis of the intersecting gene targets of diabetic kidney disease (DKD) and 3-MA was performed, and the results were verified by Western blotting both in vivo and in vitro. Results In the diabetic mice, treatment with 3-MA produced a short-term hypoglycemic effect, reduced the kidney/body weight ratio and glomerular hypertrophy, and decreased the expressions of α‑SMA and PCNA in the renal cortex. In the in vitro study, 3-MA significantly lowered the viability and reduced PCNA expression in SV40 MES 13 cells exposed to high glucose. The results of bioinformatic analysis identified AKT1 as the key gene in the therapeutic mechanism of 3-MA for DKD. Western blotting confirmed that 3-MA inhibited the phosphorylation of AKT and S6 in both the renal cortex of diabetic mice and high glucose-treated SV40 MES 13 cells. Conclusion 3-MA suppresses mesangial cell proliferation and alleviates early diabetic renal injury in mice possibly by inhibiting AKT signaling.

Key words: 3-methyladenine, diabetic kidney disease, mesangial cells, renal injury

刘本菊, 王业磊, 任海文, 欧丽雯, 邓轩, 黄梦欣, 吴鑫, 龚权. 3-甲基腺嘌呤通过抑制AKT信号减轻糖尿病小鼠的早期肾损伤[J]. 南方医科大学学报, 2024, 44(7): 1236-1242.

Benju LIU, Yelei WANG, Haiwen REN, Liwen OU, Xuan DENG, Mengxin HUANG, Xin WU, Quan GONG. 3-Methyladenine alleviates early renal injury in diabetic mice by inhibiting AKT signaling[J]. Journal of Southern Medical University, 2024, 44(7): 1236-1242.

图/表 6

图1 3-MA对DM小鼠空腹血糖和体质量的影响

Fig.1 Effects of 3-MA on fasting blood glucose and body weight of diabetic mice (Mean±SD, n=8). A: Fasting blood glucose. B: Body weight. *P<0.001 vs CON group; #P<0.001 vs DM+3-MA group.

图2 3-MA对DM小鼠肾/体质量比和肾小球大小的影响

Fig.2 Effects of 3-MA on kidney/body weight ratio and glomerular size of diabetic mice (Mean±SD, n=8). A: Kidney/body weight ratio. B: Percentage of glomerular area to field of view. C: Pepresentative images of PAS staining (Original magnification: ×400). *P<0.05, ***P<0.001 vs CON group; #P<0.05 vs DM group.

图3 3-MA对DM小鼠肾皮质α-SMA和PCNA蛋白表达的影响

Fig.3 Effects of 3-MA on the protein expressions of α-SMA and PCNA in the renal cortex of the diabetic mice (Mean±SD, n=4). A: Western blotting of α-SMA expression. B: Quantification of α-SMA protein expression. C: Western blotting of PCNA expression. D: Quantification of PCNA protein expression. E: Immunohistochemical staining of PCNA. **P<0.01 vs CON group; ##P<0.01, ###P<0.001 vs DM group.

图4 3-MA对高糖处理的SV40 MES 13细胞活力和PCNA表达的影响

Fig.4 Effects of 3-MA on cell viability and PCNA expression of SV40 MES 13 cells exposed to high glucose (Mean±SD, n=4). A: Cell viability. B: Western blotting of PCNA expression. C: Quantification of PCNA protein expression. **P<0.01, ***P<0.001 vs NG group; ###P<0.001 vs HG group.

图5 3-MA作用于DKD的生信分析

Fig.5 Bioinformatic analysis of 3-MA on diabetic kidney disease. A: Venn diagram of intersecting genes between diabetic kidney disease targets and 3-MA predicted targets. B: KEGG enrichment analysis. C: Protein-protein interaction analysis network.

图6 3-MA对DM小鼠和高糖处理的SV40 MES 13细胞AKT信号分子的影响

Fig.6 Effects of 3-MA on AKT signaling molecules in diabetic mice and high glucose treated SV40 MES 13 cells (Mean±SD, n=4). A: Western blotting of p-AKT (Ser473), AKT, p-S6, and S6 in the renal cortex of mice. B: Quantification of the ratio of p-AKT/AKT and p-S6/S6 in the renal cortex of mice. C: Western blotting of p-AKT (Ser473), AKT, p-S6, and S6 in SV40 MES 13 cells. D: Quantification of the ratio of p-AKT/AKT and p-S6/S6 in SV40 MES 13 cells. *P<0.05, **P<0.01, ***P<0.001 vs CON or NG group; #P<0.05, ##P<0.01, ###P<0.001 vs DM or HG group.

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3-甲基腺嘌呤通过抑制AKT信号减轻糖尿病小鼠的早期肾损伤

3-Methyladenine alleviates early renal injury in diabetic mice by inhibiting AKT signaling

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