Wang Yuxin, Ma Yuhua, Ke Yanrong, Jiang Xiaocheng, Liu Jian, Xiao Yang, Zheng Hong, Wang Chaojun, Chen Xue, Shi Manman
Department of Nephrology, Traditional Chinese Medicine Hospital of Kunshan, Kunshan, Jiangsu, China.
Department of Nephrology, Traditional Chinese Medicine Hospital of Kunshan, Kunshan, Jiangsu, China.
J Ethnopharmacol. 2024 Apr 24;324:117734. doi: 10.1016/j.jep.2024.117734. Epub 2024 Jan 16.
Fangji Huangqi Decoction (FJHQ), a traditional Chinese medicinal formula outlined in Zhang Zhongjing's "Jin Gui Yao Lue" during the Han Dynasty, is often used to treat conditions characterized by symptoms like edema and dysuria, including membranous nephropathy (MN). Despite its proven clinical effectiveness, the exact mechanisms through which FJHQ acts on MN remain elusive.
This study aimed to investigate whether FJHQ enhances BNIP3-mediated mitophagy in podocytes by promoting BNIP3 expression and whether this improvement leads to the amelioration of MN.
In this study, by establishing passive Heymann nephritis (PHN) rats, an experimental rat model of MN induced by sheep anti-rat Fx1A serum, we evaluated the effects of FJHQ in vivo. In vitro experiments were carried out by treating primary podocytes with experimental rat serum. Furthermore, the potential mechanism by which FJHQ acts through BNIP3 was further examined by transfecting primary podocytes with the siRNA of BNIP3 or the corresponding control vector.
After 4 weeks, significant kidney damage was observed in the rats in the model group, comparatively, FJHQ markedly decreased urine volume, 24-h urinary protein, blood urea nitrogen (BUN), creatinine (Scr), and increased serum total albumin (ALB). Histology showed that FJHQ caused significant improvements in glomerular hyperplasia, and IgG immune complex deposition in MN rats. JC-1 fluorescence labelling and flow cytometry analysis showed that FJHQ could significantly increase mitochondrial membrane potential in vivo. In the mitochondria of MN model rats, FJHQ was able to down-regulate the expression of P62 and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I, according to Western blot and immunofluorescence studies. Furthermore, FJHQ has been shown to significantly up-regulate mitochondrial membrane potential, down-regulate P62 expression in mitochondria, and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I in mitochondria at the cellular level. After the administration of the autophagy inhibitor chloroquine, the serum of rats treated with FJHQ further increased the expression of LC3 II/LC3 I in primary podocytes, showing higher autophagy flow. After the interference of BNIP3 in podocytes, the effect of FJHQ on mitochondrial membrane potential and autophagy-related proteins almost disappeared.
FJHQ enhanced mitophagy in podocytes by promoting the expression of BNIP3, thereby contributing to the amelioration of MN. This work reveals the possible underlying mechanism by which FJHQ improves MN and provides a new avenue for MN treatment.
防己黄芪汤（FJHQ）是汉代张仲景《金匮要略》中记载的一种中药方剂，常用于治疗以水肿、排尿困难等症状为特征的病症，包括膜性肾病（MN）。尽管其临床疗效已得到证实，但FJHQ作用于MN的确切机制仍不清楚。
本研究旨在探讨FJHQ是否通过促进BNIP3表达增强足细胞中BNIP3介导的线粒体自噬，以及这种改善是否导致MN病情的改善。
在本研究中，通过建立被动型Heymann肾炎（PHN）大鼠，即一种由羊抗大鼠Fx1A血清诱导的MN实验大鼠模型，我们评估了FJHQ在体内的作用。体外实验通过用实验大鼠血清处理原代足细胞进行。此外，通过用BNIP3的小干扰RNA（siRNA）或相应的对照载体转染原代足细胞，进一步研究FJHQ通过BNIP3发挥作用的潜在机制。
4周后，模型组大鼠出现明显的肾损伤，相比之下，FJHQ显著降低尿量、24小时尿蛋白、血尿素氮（BUN）、肌酐（Scr），并增加血清总白蛋白（ALB）。组织学显示，FJHQ使MN大鼠的肾小球增生和IgG免疫复合物沉积得到显著改善。JC-1荧光标记和流式细胞术分析表明，FJHQ能显著提高体内线粒体膜电位。根据蛋白质免疫印迹和免疫荧光研究，在MN模型大鼠的线粒体中，FJHQ能够下调P62的表达，并上调BNIP3、LC3B和LC3 II/LC3 I的表达。此外，在细胞水平上，FJHQ已被证明能显著上调线粒体膜电位，下调线粒体中P62的表达，并上调线粒体中BNIP3、LC3B和LC3 II/LC3 I的表达。给予自噬抑制剂氯喹后，FJHQ处理的大鼠血清进一步增加了原代足细胞中LC3 II/LC3 I的表达，显示出自噬流更高。在足细胞中干扰BNIP3后，FJHQ对线粒体膜电位和自噬相关蛋白的作用几乎消失。
FJHQ通过促进BNIP3表达增强足细胞中的线粒体自噬，从而有助于改善MN。这项工作揭示了FJHQ改善MN的可能潜在机制，并为MN治疗提供了一条新途径。
