帕立骨化醇通过抑制 MAPK、Akt 和 NF-κB 的激活来减轻 HK-2 细胞中吲哚硫酸酯诱导的细胞凋亡。
Paricalcitol attenuates indoxyl sulfate-induced apoptosis through the inhibition of MAPK, Akt, and NF-kB activation in HK-2 cells.
机构信息
Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.
出版信息
Korean J Intern Med. 2019 Jan;34(1):146-155. doi: 10.3904/kjim.2016.298. Epub 2017 Oct 10.
BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury.
METHODS
The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF- κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein.
RESULTS
IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells.
CONCLUSION
Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.
背景/目的：硫酸吲哚酚（IS）是一种尿毒症毒素，也是慢性肾脏病进展的重要致病因素。最近，帕立骨化醇（19-去甲-1,25-二羟维生素 D2）已被证明对肾损伤具有保护作用。在此，我们研究了帕立骨化醇治疗对 IS 诱导的肾小管损伤的影响。
方法
用荧光染料 2',7'-二氯荧光素二乙酸酯测量 IS 给药后人肾近端小管上皮（HK-2）细胞内的活性氧（ROS）。用 MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐）测定 IS 对细胞活力的影响，并用半定量免疫印迹法测定凋亡相关蛋白（Bcl-2 相关蛋白 X [Bax]和 B 细胞淋巴瘤 2 [Bcl-2]）、核因子-κB（NF- κB）p65、丝裂原激活蛋白激酶（MAPK）和蛋白激酶 B（Akt）的磷酸化水平。通过荧光素酶测定法测量 NF-κB 的启动子活性，并用异硫氰酸荧光素结合的 Annexin V 蛋白染色的细胞流式细胞术测定细胞凋亡。
结果
IS 处理增加了 ROS 的产生，降低了 HK-2 细胞的活力并诱导了细胞凋亡。IS 处理增加了凋亡相关蛋白 Bax 的表达，降低了 Bcl-2 的表达，并激活了 MAPK、NF-κB p65 和 Akt 的磷酸化。相反，帕立骨化醇处理降低了 Bax 的表达，增加了 Bcl-2 的表达，并抑制了 MAPK、NF-κB p65 和 Akt 的磷酸化。IS 给药后 NF-κB 启动子活性增加，并用帕立骨化醇预处理拮抗。此外，流式细胞术分析表明，IS 诱导的凋亡被帕立骨化醇处理减弱，导致异硫氰酸荧光素结合的 Annexin V 阳性细胞数量减少。
结论
帕立骨化醇通过调节 MAPK、NF-κB 和 Akt 信号通路抑制 HK-2 细胞中 IS 诱导的凋亡。
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