本研究旨在探讨参芪地黄汤对高糖诱导的人肾小管上皮细胞(HK-2)铁性凋亡及核因子红细胞相关因子2(Nrf2)/血红素加氧酶-1(HO-1)/谷胱甘肽过氧化物酶4(GPX4)轴的影响,并阐明其作用机制。通过将HK-2暴露于高糖,建立细胞损伤模型。制备参芪地黄汤含药血清。确定参芪地黄汤的最佳浓度和干预时间。HK-2分为正常,高葡萄糖,和低,medium-,参芪地黄汤高剂量组。干预后,测定各组细胞增殖率,观察细胞形态和线粒体超微结构。然后,细胞内活性氧(ROS)的水平,亚铁离子(Fe~(2+)),谷胱甘肽(GSH),测定丙二醛(MDA)和Nrf2、HO-1、GPX4和xCT蛋白水平。确定参芪地黄汤含药血清的最佳浓度和干预时间为10%和24h。分别。与高糖组相比,大剂量参芪地黄汤促进HK-2的增殖。细胞在低,medium-,参芪地黄汤高剂量组排列紧密,细胞计数增加,从纺锤体纤维形状到鹅卵石形状的改进形态,线粒体膜和cr的形态和结构得到改善,与高糖组相比。同时,所有剂量的参芪地黄汤都能抑制ROS的升高,以减轻过氧化损伤,降低Fe~(2+)和MDA水平,升高GSH水平,抑制脂质过氧化,并激活抗氧化途径上调Nrf2,HO-1,xCT,GPX4。总之,参芪地黄汤含药血清能抑制高糖诱导的HK-2细胞凋亡,其中涉及抗氧化作用和Nrf2/HO-1/GPX4途径的激活。
This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.