Quan Xiaohong, Liu Huihui, Ye Dongmei, Ding Xinling, Su Xiulan
Experiment & Teaching Center for Basic Medicine, Chifeng University School of Basic Medical Sciences, Chifeng, Inner Mongolia, 024000, People's Republic of China.
Core Facility Center for Functional Experiments, CUSBMS, Chifeng University School of Basic Medical Sciences, Chifeng, Inner Mongolia, 024000, People's Republic of China.
Diabetes Metab Syndr Obes. 2021 Apr 28;14:1885-1895. doi: 10.2147/DMSO.S305092. eCollection 2021.
Podocyte injury serves an important role during the progression of diabetic nephropathy (DN). The aim of this study was to investigate the effects of forsythoside A (FA) on high glucose (HG)-induced podocyte injury and to identify the possible mechanisms.
MPC-5 podocytes were cultured under HG conditions. After exposure to different doses of FA, cell viability and apoptosis were respectively evaluated with CCK-8 assay and flow cytometry. Then, the levels of oxidative stress-related markers and inflammatory factors were examined by corresponding kits. Western blot analysis was employed to detect the expression of Nox2, Nox4, COX-2, iNOS and matrix metalloproteinases 12 (MMP12). Subsequently, MMP12 was overexpressed to assess whether the effects of FA on HG-stimulated podocyte injury were mediated by MMP12 and MAPK signaling.
Results indicated that FA dose-dependently elevated cell viability, reduced cell apoptosis in HG-induced MPC-5 cells. Additionally, FA significantly inhibited oxidative stress, which could be certified by decreased content of malondialdehyde (MDA), enhanced activities of superoxide dismutase (SOD) and catalase (CAT), and downregulated expression of Nox2 and Nox4. Moreover, notably reduced levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were observed in FA-treated MPC-5 cells under HG conditions, accompanied by decreased COX-2 and iNOS expression. Remarkably, FA suppressed MMP12 expression in a dose-dependent manner, and the effects of FA on MPC-5 cells exposed to HG were partially counteracted by MMP12 overexpression. Mechanically, FA inactivated the expression of phospho-ERK (p-ERK), p-p38 and p-JNK, which was restored after MMP12 overexpression.
These findings demonstrate a protective mechanism of FA by inactivating MAPK signaling via MMP12 inhibition in HG-induced podocyte injury, providing a promising therapeutic candidate for the treatment of DN.
足细胞损伤在糖尿病肾病（DN）进展过程中起重要作用。本研究旨在探讨连翘酯苷A（FA）对高糖（HG）诱导的足细胞损伤的影响，并确定其可能的机制。
将MPC-5足细胞在HG条件下培养。暴露于不同剂量的FA后，分别用CCK-8法和流式细胞术评估细胞活力和凋亡情况。然后，通过相应试剂盒检测氧化应激相关标志物和炎症因子的水平。采用蛋白质印迹法检测Nox2、Nox4、COX-2、iNOS和基质金属蛋白酶12（MMP12）的表达。随后，过表达MMP12以评估FA对HG刺激的足细胞损伤的影响是否由MMP12和MAPK信号介导。
结果表明，FA剂量依赖性地提高HG诱导的MPC-5细胞的活力，降低细胞凋亡。此外，FA显著抑制氧化应激，这可通过丙二醛（MDA）含量降低、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性增强以及Nox2和Nox4表达下调得到证实。此外，在HG条件下，FA处理的MPC-5细胞中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β和IL-6水平显著降低，同时COX-2和iNOS表达减少。值得注意的是，FA以剂量依赖性方式抑制MMP12表达，MMP12过表达部分抵消了FA对暴露于HG的MPC-5细胞的影响。机制上，FA使磷酸化细胞外信号调节激酶（p-ERK）、磷酸化p38和磷酸化c-Jun氨基末端激酶（p-JNK）的表达失活，MMP12过表达后恢复。
这些发现证明了FA通过在HG诱导的足细胞损伤中抑制MMP12使MAPK信号失活的保护机制，为DN的治疗提供了一个有前景的治疗候选药物。
