Department of Nephrology, The Third Affiliated Hospital of Qiqihar Medical University, Qiqihar, Heilongjiang, China.
Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar, Heilongjiang, China.
Clin Hemorheol Microcirc. 2024;86(3):369-382. doi: 10.3233/CH-231979.
To investigate the mechanism by which NF-κB p65 activates miR-150 to suppress TRPC6 expression and promote renal ischemia-reperfusion injury.
To assess the transcription of miR-150, NF-B p65, and TRPC6 in HK-2 cells treated with hypoxia reperfusion and rat kidney tissue damaged by ischemia-reperfusion (I/R), qPCR was implemented. The protein production of NF-κB p65 and TRPC6 was assessed by Western blot (WB) analysis. The histological score of rat kidney tissue was assessed using H&E (hematoxylin and eosin) staining. To assess the rate of apoptosis of renal tissue cells following I/R injury, we used the TACS TdT In Situ Apoptosis Detection Kit. To find out the impairment of renal function, blood levels of creatinine (Cr) and blood urea nitrogen (BUN) were tested in rats. Concentrations of inflammatory cytokines, including IL-1β, IL-10, and TNF-α, were detected in HK-2 cells and rat renal tissue cells utilizing ELISA kits. FITC and CCK-8 were employed to analyze the death rate and cellular proliferation of HK-2 cells. To analyse the mechanism of engagement between NF-κB p65 and the miR-150 promoter, coupled with the detrimental impact of miR-150 on TRPC6, we adopted the dual-luciferase reporter assay. To confirm the activating effect of NF-κB p65 on miR-150,we implemented the ChIP assay.
NF-κB p65 expression was significantly upregulated in rat renal tissue following IRI. Applying the dual-luciferase reporter assay, we demonstrated that the specific attachment of NF-B p65 with the miR-150 promoter location is viable, resulting in the promotion of the activity of the promoter. When miR-150 was overexpressed, we observed a notable reduction in cell proliferation. And it notably increased the rate of cellular apoptosis rate and amounts of the proinflammatory cytokines IL-1β, IL-10, and TNF-α. Employing the dual-luciferase reporter assay, we demonstrated that miR-150 transfection diminished the function of luciferase in the TRPC6-WT group, whereas luciferase activity in the TRPC6-MUT group remained unchanged, indicating that miR-150 is a targeted inhibitor of TRPC6. In the rat renal I/R model, when miR-150 was inhibited or TRPC6 was overexpressed in the rat kidney I/R model, the histological score of rat kidney tissue significantly decreased, so did the quantities of proinflammatory cytokines IL-1β, IL-10, TNF-α, creatinine (Cr) and blood urea nitrogen (BUN) contents and the rate of cell apoptosis in kidney tissue.
Activation of miR-150 by NF-κB p65 results in downregulation of TRPC6 expression and promotion of IRI in the kidney.
研究 NF-κB p65 如何激活 miR-150 抑制 TRPC6 表达并促进肾缺血再灌注损伤。
采用 qPCR 检测缺氧再灌注处理的 HK-2 细胞和缺血再灌注（I/R）损伤大鼠肾组织中 miR-150、NF-B p65 和 TRPC6 的转录，采用 Western blot（WB）分析检测 NF-κB p65 和 TRPC6 的蛋白产生，采用 H&E 染色评估大鼠肾组织的组织学评分。使用 TACS TdT 原位细胞凋亡检测试剂盒评估 I/R 损伤后肾组织细胞的凋亡率。检测大鼠血肌酐（Cr）和血尿素氮（BUN）水平，以评估肾功能损伤。采用 ELISA 试剂盒检测 HK-2 细胞和大鼠肾组织细胞中炎症细胞因子 IL-1β、IL-10 和 TNF-α 的浓度。采用 FITC 和 CCK-8 分析 HK-2 细胞的死亡率和细胞增殖。采用双荧光素酶报告基因检测分析 NF-κB p65 与 miR-150 启动子之间的结合机制及其对 TRPC6 的损害作用，采用 ChIP assay 验证 NF-κB p65 对 miR-150 的激活作用。
I/R 后大鼠肾组织中 NF-κB p65 的表达明显上调。通过双荧光素酶报告基因检测，我们证明了 NF-B p65 与 miR-150 启动子位置的特异性结合是可行的，这导致了启动子活性的增强。当 miR-150 过表达时，我们观察到细胞增殖明显减少。同时，细胞凋亡率显著增加，促炎细胞因子 IL-1β、IL-10 和 TNF-α 的含量也显著增加。通过双荧光素酶报告基因检测，我们发现 miR-150 转染降低了 TRPC6-WT 组中荧光素酶的功能，而 TRPC6-MUT 组中荧光素酶活性保持不变，表明 miR-150 是 TRPC6 的靶向抑制剂。在大鼠肾 I/R 模型中，当在大鼠肾 I/R 模型中抑制 miR-150 或过表达 TRPC6 时，大鼠肾组织的组织学评分显著降低，大鼠肾组织中促炎细胞因子 IL-1β、IL-10、TNF-α、肌酐（Cr）和血尿素氮（BUN）含量以及肾组织细胞凋亡率也显著降低。
NF-κB p65 激活 miR-150 导致肾组织中 TRPC6 表达下调并促进 IRI。
