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Abstract：
BACKGROUND: Diabetic nephropathy is a major secondary cause of end-stage renal disease. Apelin plays an important role in the development of DN. Understanding the exact mechanism of Apelin can help expand the means of treating DN.
METHODS: Male C57BL/6 mice was used and STZ treatment was implemented for DN model establishment. Lentivirus systems including Lv-sh-RUNX3 and Lv-Apelin were obtained to knockdown RUNX3 and overexpress Apelin, respectively. A total of 36 mice were divided into 6 groups (n = 6 in each group): control, DN, DN + LV-Vector, DN + Lv-Apelin, DN + LV-Apelin + LV-sh-NC and DN + Lv-Apelin + Lv-sh-RUNX3 group. In vitro studies were performed using mesangial cells. Cell viability and proliferation was assessed through CCK8 and EDU analysis. Hematoxylin and eosin staining as well as Masson staining was implemented for histological evaluation. RT-qPCR was conducted for measuring relative mRNA levels, and protein expression was detected by western blotting. The interaction between SIRT1 and FOXO were verified by co-immunoprecipitations, and relations between RUNX3 and Apelin were demonstrated by dual luciferase report and chromatin immunoprecipitation.
RESULTS: The DN group exhibited significantly lower Apelin expression compared to control (p < 0.05). Apelin overexpression markedly improved blood glucose, renal function indicators, ameliorated renal fibrosis and reduced fibrotic factor expression (p < 0.05) in the DN group, accompanied by elevated sirt1 levels and diminished acetylated FOXO1/FOXO3a (p < 0.05). However, RUNX3 knockdown combined with Apelin overexpression abrogated these beneficial effects, leading to impaired renal function, exacerbated fibrosis, increased fibrotic factor expression and acetylated FOXO1/FOXO3a versus Apelin overexpression alone (p < 0.05). In mesangial cells under high glucose, Apelin overexpression significantly inhibited cell proliferation and fibrotic factor production (p < 0.05). Conversely, RUNX3 interference enhanced cell proliferation and the secretion of fibrotic factors. (p < 0.05). Remarkably, combining Apelin overexpression with RUNX3 interference reversed the proliferation and fibrosis induced by RUNX3 interference (p < 0.05). Mechanistic studies revealed RUNX3 binds to the Apelin promoter, with the 467-489 bp site1 as the primary binding region, and SIRT1 physically interacts with FOXO1 and FOXO3a in mesangial cells.
CONCLUSIONS: RUNX3 activated Apelin and regulated the SIRT1/FOXO signaling pathway, resulting in the suppressed cell proliferation and fibrosis in diabetic nephropathy. Apelin is a promising endogenous therapeutic target for anti-renal injury and anti-fibrosis in diabetic nephropathy. RUNX3 may serve as an endogenous intervention target for diseases related to Apelin deficiency.
METHODS: Male C57BL/6 mice was used and STZ treatment was implemented for DN model establishment. Lentivirus systems including Lv-sh-RUNX3 and Lv-Apelin were obtained to knockdown RUNX3 and overexpress Apelin, respectively. A total of 36 mice were divided into 6 groups (n = 6 in each group): control, DN, DN + LV-Vector, DN + Lv-Apelin, DN + LV-Apelin + LV-sh-NC and DN + Lv-Apelin + Lv-sh-RUNX3 group. In vitro studies were performed using mesangial cells. Cell viability and proliferation was assessed through CCK8 and EDU analysis. Hematoxylin and eosin staining as well as Masson staining was implemented for histological evaluation. RT-qPCR was conducted for measuring relative mRNA levels, and protein expression was detected by western blotting. The interaction between SIRT1 and FOXO were verified by co-immunoprecipitations, and relations between RUNX3 and Apelin were demonstrated by dual luciferase report and chromatin immunoprecipitation.
RESULTS: The DN group exhibited significantly lower Apelin expression compared to control (p < 0.05). Apelin overexpression markedly improved blood glucose, renal function indicators, ameliorated renal fibrosis and reduced fibrotic factor expression (p < 0.05) in the DN group, accompanied by elevated sirt1 levels and diminished acetylated FOXO1/FOXO3a (p < 0.05). However, RUNX3 knockdown combined with Apelin overexpression abrogated these beneficial effects, leading to impaired renal function, exacerbated fibrosis, increased fibrotic factor expression and acetylated FOXO1/FOXO3a versus Apelin overexpression alone (p < 0.05). In mesangial cells under high glucose, Apelin overexpression significantly inhibited cell proliferation and fibrotic factor production (p < 0.05). Conversely, RUNX3 interference enhanced cell proliferation and the secretion of fibrotic factors. (p < 0.05). Remarkably, combining Apelin overexpression with RUNX3 interference reversed the proliferation and fibrosis induced by RUNX3 interference (p < 0.05). Mechanistic studies revealed RUNX3 binds to the Apelin promoter, with the 467-489 bp site1 as the primary binding region, and SIRT1 physically interacts with FOXO1 and FOXO3a in mesangial cells.
CONCLUSIONS: RUNX3 activated Apelin and regulated the SIRT1/FOXO signaling pathway, resulting in the suppressed cell proliferation and fibrosis in diabetic nephropathy. Apelin is a promising endogenous therapeutic target for anti-renal injury and anti-fibrosis in diabetic nephropathy. RUNX3 may serve as an endogenous intervention target for diseases related to Apelin deficiency.
摘要：
背景：糖尿病肾病是终末期肾病的主要次要原因。Apelin在DN的发展中起着重要作用。了解Apelin的确切机制有助于拓展治疗DN的手段。
方法：采用雄性C57BL/6小鼠，采用STZ处理建立DN模型。获得包括Lv-sh-RUNX3和Lv-Apelin的慢病毒系统以敲低RUNX3并过表达Apelin，分别。将36只小鼠分为6组(每组n=6):对照组,DN,DN+LV-Vector,DN+Lv-Apelin,DN+LV-Apelin+LV-sh-NC和DN+Lv-Apelin+Lv-sh-RUNX3组。使用系膜细胞进行体外研究。通过CCK8和EDU分析评估细胞活力和增殖。实施苏木精和伊红染色以及Masson染色用于组织学评价。进行RT-qPCR以测量相对mRNA水平,蛋白质印迹法检测蛋白质表达。SIRT1和FOXO之间的相互作用通过免疫共沉淀进行验证，通过双荧光素酶报告和染色质免疫沉淀证明了RUNX3和Apelin之间的关系。
结果：与对照组相比，DN组的Apelin表达明显降低（p<0.05）。Apelin过表达显著改善血糖，肾功能指标,DN组肾脏纤维化改善，纤维化因子表达降低（p<0.05），伴有sirt1水平升高和乙酰化FOXO1/FOXO3a减少（p<0.05）。然而,RUNX3敲低结合Apelin过表达消除了这些有益作用，导致肾功能受损，加剧纤维化，与单独的Apelin过表达相比，纤维化因子表达和乙酰化FOXO1/FOXO3a增加（p<0.05）。在高葡萄糖下的肾小球膜细胞中，Apelin过表达显著抑制细胞增殖和纤维化因子产生(p<0.05)。相反，RUNX3干扰增强细胞增殖和纤维化因子的分泌。(p<0.05)。值得注意的是,Apelin过表达与RUNX3干扰联合逆转了RUNX3干扰诱导的增殖和纤维化(p<0.05)。机制研究显示RUNX3与Apelin启动子结合,以467-489bp的位点1为主要结合区，SIRT1与肾小球系膜细胞中的FOXO1和FOXO3a发生物理相互作用。
结论：RUNX3激活Apelin并调节SIRT1/FOXO信号通路，导致糖尿病肾病细胞增殖抑制和纤维化。Apelin是糖尿病肾病中抗肾损伤和抗纤维化的有前景的内源性治疗靶点。RUNX3可作为Apelin缺乏相关疾病的内源性干预靶点。
方法：采用雄性C57BL/6小鼠，采用STZ处理建立DN模型。获得包括Lv-sh-RUNX3和Lv-Apelin的慢病毒系统以敲低RUNX3并过表达Apelin，分别。将36只小鼠分为6组(每组n=6):对照组,DN,DN+LV-Vector,DN+Lv-Apelin,DN+LV-Apelin+LV-sh-NC和DN+Lv-Apelin+Lv-sh-RUNX3组。使用系膜细胞进行体外研究。通过CCK8和EDU分析评估细胞活力和增殖。实施苏木精和伊红染色以及Masson染色用于组织学评价。进行RT-qPCR以测量相对mRNA水平,蛋白质印迹法检测蛋白质表达。SIRT1和FOXO之间的相互作用通过免疫共沉淀进行验证，通过双荧光素酶报告和染色质免疫沉淀证明了RUNX3和Apelin之间的关系。
结果：与对照组相比，DN组的Apelin表达明显降低（p<0.05）。Apelin过表达显著改善血糖，肾功能指标,DN组肾脏纤维化改善，纤维化因子表达降低（p<0.05），伴有sirt1水平升高和乙酰化FOXO1/FOXO3a减少（p<0.05）。然而,RUNX3敲低结合Apelin过表达消除了这些有益作用，导致肾功能受损，加剧纤维化，与单独的Apelin过表达相比，纤维化因子表达和乙酰化FOXO1/FOXO3a增加（p<0.05）。在高葡萄糖下的肾小球膜细胞中，Apelin过表达显著抑制细胞增殖和纤维化因子产生(p<0.05)。相反，RUNX3干扰增强细胞增殖和纤维化因子的分泌。(p<0.05)。值得注意的是,Apelin过表达与RUNX3干扰联合逆转了RUNX3干扰诱导的增殖和纤维化(p<0.05)。机制研究显示RUNX3与Apelin启动子结合,以467-489bp的位点1为主要结合区，SIRT1与肾小球系膜细胞中的FOXO1和FOXO3a发生物理相互作用。
结论：RUNX3激活Apelin并调节SIRT1/FOXO信号通路，导致糖尿病肾病细胞增殖抑制和纤维化。Apelin是糖尿病肾病中抗肾损伤和抗纤维化的有前景的内源性治疗靶点。RUNX3可作为Apelin缺乏相关疾病的内源性干预靶点。
