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| Sirt1在急性炎症状态下糖尿病小鼠肾损伤中的作用 |
| Sirt1 regulates cell scorch pathway to protect renal function in diabetic mice under acute inflammatory state |
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| DOI：10.12089/jca.2023.12.012 |
| 中文关键词: 糖尿病 急性肾损伤 焦亡 沉默信息调节因子1 乙酰化 叉头状转录调节因子3a |
| 英文关键词: Diabetes Kidney injury Pyroptosis Sirt 1 protein Acetylated FoxO3a protein |
| 基金项目:大学生创新创业训练计划（202110660007）；贵州省卫健委科学技术基金（gzwkj2022-124） |
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| 中文摘要: |
目的 探究Sirt1在急性炎症状态下糖尿病小鼠肾损伤中的作用及分子机制。
方法 选择SPF级C57BL/6J雄性小鼠40只，8周龄，体重20～25 g。采用随机数字表法将小鼠分为五组：对照组（C组）、糖尿病组（D组）、脂多糖（LPS）+糖尿病组（L组）、LPS+糖尿病+Sirt1阻断剂EX527组（E组）和LPS+糖尿病+Sirt1激动剂银杏黄酮苷元组（G组），每组8只。糖尿病小鼠模型制备成功后，L组腹腔注射LPS 10 mg/kg；E组在糖尿病小鼠给予LPS处理前1 h腹腔注射EX527 5 mg/kg（溶于DMSO 0.2 ml）；G组在糖尿病小鼠给予LPS处理前1 h腹腔注射银杏黄酮苷元200 mg/kg（溶于DMSO 0.2 ml）；C组和D组在相同时点腹腔注射2%DMSO 0.15 ml。收集24 h尿液测定24 h尿量和24 h尿蛋白浓度，眼球取血检测血清肌酐（Scr）和尿素氮（BUN）浓度。取肾组织后采用ELISA法检测IL-1β、IL-18浓度，硝酸还原酶法检测一氧化氮（NO）含量，铁离子抗氧化能力法检测总抗氧能力（T-AOC），qPCR和Western blot法检测Sirt1、caspase-1、NLRP3、ASC mRNA表达量和蛋白含量，免疫共沉淀法检测乙酰化FoxO3a蛋白含量，二氢乙锭染色法计算活性氧（ROS）含量，免疫荧光双重染色法计算细胞焦亡率，并进行HE染色，光镜下观察病理。
结果 与C组比较，D组、L组、E组和G组24 h尿量明显增多，尿蛋白浓度、血清Scr和BUN浓度、肾组织IL-1β、IL-18、NO含量、NLRP3、caspase-1、ASC mRNA表达量和蛋白含量、ROS含量和焦亡率均明显升高（P<0.05），T-AOC活力明显降低（P<0.05）。与D组比较，L组、E组和G组24 h尿量明显增多，尿蛋白浓度、血清Scr和BUN浓度、肾组织IL-1β、IL-18、NO含量、NLRP3、caspase-1、ASC mRNA表达量和蛋白含量、ROS含量和焦亡率均明显升高（P<0.05），T-AOC活力明显降低（P<0.05）。与L组比较，E组24 h尿量明显增多，尿蛋白浓度、血清Scr和BUN浓度、肾组织IL-1β、IL-18、NO含量、NLRP3、caspase-1、ASC mRNA表达量和蛋白含量、乙酰化FoxO3a蛋白含量、ROS含量和焦亡率均明显升高（P<0.05），T-AOC活力、Sirt1 mRNA和蛋白含量、FoxO3a mRNA表达量均明显降低（P<0.05）；G组24 h尿量明显减少，尿蛋白浓度、血清Scr和BUN浓度、肾组织IL-1β、IL-18、NO含量、NLRP3、caspase-1、ASC mRNA表达量和蛋白含量、乙酰化FoxO3a蛋白含量、ROS含量和焦亡率均明显降低（P<0.05），T-AOC活力、Sirt1 mRNA表达量和蛋白含量均明显升高（P<0.05）。与E组比较，G组24 h尿量明显减少，尿蛋白浓度、血清Scr和BUN浓度、肾组织IL-1β、IL-18、NO浓度、NLRP3、caspase-1和ASC mRNA表达量和蛋白含量、乙酰化FoxO3a蛋白含量、ROS含量和细胞焦亡率均明显降低（P<0.05），T-AOC活力、Sirt1 mRNA表达量和蛋白含量均明显升高（P<0.05）。
结论 在急性炎症状态下的糖尿病小鼠中，Sirt1升高可以降低乙酰化FoxO3a蛋白含量，减少小鼠尿量，降低尿蛋白、Scr、BUN含量、肾组织中炎性因子浓度和细胞焦亡水平，减轻氧化应激和炎症水平，从而减轻肾损伤。 |
| 英文摘要: |
Objective To investigate the role and molecular mechanism of Sirt1 in renal injury in diabetic mice under acute inflammatory state.
Methods Forty SPF grade C57BL/6J male mice, 8 weeks old, weighing 20-25 g were selected. The mice were divided into five groups by random number table method: control group (group C), diabetic group (group D), lipopolysaccharide (LPS) + diabetic group (group L), LPS + diabetic + Sirt1 blocker EX527 group (group E), and LPS + diabetic + Sirt1 agonist ginkgoflavone sapogenins group (group G), 8 mice in each group. After successful preparation of the diabetic mouse model, group L was injected intraperitoneally with LPS 10 mg/kg. Group E was injected intraperitoneally with EX527 5 mg/kg (dissolved in DMSO 0.2 ml) 1 hour before giving LPS treatment to diabetic mice. Group G was injected intraperitoneally with 200 mg/kg of ginkgoflavone sapogenins (dissolved in DMSO 0.2 ml) 1 hour before LPS treatment was given to diabetic mice, groups C and D underwent an intraperitoneal injection of 2% DMSO 0.15 ml at the same time point. 24-hours urine volume was collected and 24-hours urinary protein concentration was determined, and blood was taken from the posterior eyes to detect serum Scr and BUN concentrations. After kidney tissues were removed, IL-1β and IL-18 concentrations were measured by ELISA, nitrate reductase assay for nitric oxide (NO) content in kidney, iron ion antioxidant capacity assay for total antioxidant capacity (T-AOC), qPCR and Western blot assay for Sirt1, caspase-1, NLRP3, and ASC mRNA expression and protein content. The acetylated FoxO3a protein content was detected by immunoprecipitation, the reactive oxygen species (ROS) content was calculated by dihydroethidium staining, the pyroptosis rate was calculated by immunofluorescence double staining, HE staining was performed, and the pathological results were observed under light microscope.
Results Compared with group C, 24-hours urine volume, urine protein concentration, serum Scr and BUN concentration, concentrations of renal tissue IL-1β, IL-18, and NO, NLRP3, caspase-1, and ASC mRNA expressions and protein contents, ROS content and pyroptosis rate were significantly increased, T-AOC activity was significantly decreased in groups D, L, E, and G (P < 0.05). Compared with group D, 24-hours urine volume, urine protein concentration, serum Scr and BUN concentration, concentrations of renal tissue IL-1β, IL-18, and NO, NLRP3, caspase-1, and ASC mRNA expressions and protein contents, ROS content and pyroptosis rate were significantly increased, T-AOC activity was significantly decreased in groups L, E, and G (P < 0.05). Compared with group L, 24-hours urine volume, urine protein concentration, serum Scr and BUN concentration, concentrations of renal tissue IL-1β, IL-18, and NO, NLRP3, caspase-1, and ASC mRNA expressions and protein contents, acetylated FoxO3a protein content, ROS content, and pyroptosis rate were significantly increased, T-AOC activity, Sirt1 mRNA expression and protein content, and FoxO3a mRNA expression were significantly decreased in group E (P < 0.05), 24-hours urine volume, urine protein concentration, serum Scr and BUN concentration, concentrations of renal tissue IL-1β, IL-18, and NO, NLRP3, caspase-1, and ASC mRNA expressions and protein contents, acetylated FoxO3a protein content, ROS content and pyroptosis rate were significantly decreased, T-AOC activity, Sirt1 mRNA expression and protein content were significantly increased in group G (P < 0.05). Compared with group E, 24-hours urine volume, urinary protein concentration, serum Scr and BUN concentration, concentrations of renal tissue IL-1β, IL-18, and NO, NLRP3, caspase-1, and ASC mRNA expressions and protein contents, acetylated FoxO3a protein content, ROS content, and pyroptosis rate were significantly decreased, T-AOC activity, Sirt1 mRNA expression and protein content were significantly increased in group G (P < 0.05).
Conclusion In diabetic mice under acute inflammatory state, elevated Sirt1 reduces kidney injury by decreasing acetylated FoxO3a protein content, reduced urine volume, urine protein concentration, serum Scr and BUN concentration, inflammatory factor concentrations and apoptosis levels in renal tissue, and attenuated oxidative stress and inflammation levels． |
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