Department of Nephrology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310000, China.
J Tradit Chin Med. 2019 Jun;39(3):346-355.
To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN), as well as the mechanism of action of Jixuecao (Herba Centellae Asiaticae, HCA) in the treatment of IgAN.
Adenovirus-mediated Mfn2 gene transfection and Mfn2 expression were analyzed by real-time polymerase chain reaction (PCR) and Western blotting. IgA1 induced the proliferation of GMCs, which were then treated with HCA. Cell proliferation was detected with cell counting kit-8 (CCK-8), and Mfn2 expression was analyzed by real-time PCR and western blotting. An IgAN animal model was also established and treated with HCA. GMCs proliferation was detected by hematoxylin-eosin staining, mitochondrial structure was analyzed by electron microscopy, mitochondrial function was determined by the Clark oxygen electrode method, and the expression of Mfn2, Phospho-extracellular regulated protein kinases1/2 (P-ERK1/2), Cyclin-dependent kinase 2 (CDK2), Phospho-p27 (p-p27), and cyclin A was analyzed by Western blotting.
In vitro, HCA inhibited GMCs in a concentration-dependent manner in association with the upregulation of Mfn2 expression. The overexpression of Mfn2 inhibited IgA1-induced GMCs proliferation and elevated the effect of HCA. In vivo, treatment with HCA could alleviate albuminuria and creatinine and GMCs proliferation. These effects were related to the upregulation of Mfn2, p-p27 and inhibition of p-ERK1/2, CDK2, and cyclinA. Mitochondrial swelling, vacuolar degeneration, and reduction of respiratory control rate were identified in IgAN, but HCA could improve the mitochondrial structure and function.
HCA inhibited GMCs proliferation via the upregulation Mfn2 and the inhibition of Ras-Raf-ERK/MAPK. We revealed that changes of mitochondrial structure and function are associated with IgAN, but that HCA can improve these mitochondrial features.
研究线粒体融合蛋白 2（Mfn2）及其下游信号通路对 IgA 肾病（IgAN）肾小球系膜细胞（GMC）增殖的影响，并探讨积雪草（Herba Centellae Asiaticae，HCA）治疗 IgAN 的作用机制。
通过实时聚合酶链反应（PCR）和 Western blot 分析腺病毒介导的 Mfn2 基因转染和 Mfn2 表达。用 IgA1 诱导 GMC 增殖，然后用 HCA 处理。用细胞计数试剂盒-8（CCK-8）检测细胞增殖，用实时 PCR 和 Western blot 分析 Mfn2 表达。还建立了 IgAN 动物模型，并用 HCA 进行治疗。用苏木精-伊红染色检测 GMC 增殖，用电镜分析线粒体结构，用 Clark 氧电极法测定线粒体功能，用 Western blot 分析 Mfn2、磷酸化细胞外调节蛋白激酶 1/2（P-ERK1/2）、周期蛋白依赖性激酶 2（CDK2）、磷酸化 p27（p-p27）和周期蛋白 A 的表达。
体外，HCA 呈浓度依赖性抑制 GMC，同时上调 Mfn2 表达。Mfn2 的过表达抑制 IgA1 诱导的 GMC 增殖，并增强 HCA 的作用。体内，HCA 治疗可减轻蛋白尿和肌酐以及 GMC 增殖。这些作用与 Mfn2、p-p27 的上调以及 Ras-Raf-ERK/MAPK 的抑制有关。在 IgAN 中观察到线粒体肿胀、空泡变性和呼吸控制率降低，但 HCA 可改善线粒体结构和功能。
HCA 通过上调 Mfn2 和抑制 Ras-Raf-ERK/MAPK 抑制 GMC 增殖。我们揭示了线粒体结构和功能的变化与 IgAN 有关，但 HCA 可以改善这些线粒体特征。
