Mechanism by which astragalus-peach kernel alleviates renal fibrosis in chronic kidney disease rats

doi:10.12307/2026.416

Abstract

Abstract: BACKGROUND: Our previous studies have indicated that astragalus–peach kernel alleviates the progression of chronic kidney disease yet its precise mechanism remains to be elucidated.
OBJECTIVE: To investigate the therapeutic effects and underlying mechanisms of astragalus-peach kernel in preventing renal fibrosis in chronic kidney disease rats.
METHODS: (1) Gene expression profile chip datasets related to chronic kidney disease were retrieved and filtered via the Gene Expression Omnibus (GEO) database (this public gene expression database, established and maintained by the National Center for Biotechnology Information, which is primarily used for storing and sharing high-throughput genomic data. The database offers free download and analysis tools, serves as an open-access resource, and has obtained approval from relevant institutional review boards for its research). Disease-related core targets were screened via the GEO database combined with network pharmacology analyses. Key genes obtained were validated through molecular docking. (2) Forty Sprague-Dawley rats were randomly assigned into blank, model, dapagliflozin, and astragalus–peach kernel groups (n=10 each). A chronic kidney disease model was established by intragastric administration of a 2% adenine solution in the latter three groups. Starting the day after the induction of chronic kidney disease, the blank and model groups received saline via gastric lavage, while the dapagliflozin group received dapagliflozin via gastric lavage, and the astragalus–peach kernel group received astragalus–peach kernel (with a mass ratio of 1:1) via gastric lavage, once daily for 8 consecutive weeks. Following the final administration, serum creatinine and blood urea nitrogen levels in rat arterial blood were measured. Renal tissue was examined via hematoxylin-eosin staining, Masson staining, and immunohistochemical staining for α-smooth muscle actin and type I collagen. RT-qPCR was used to detect mRNA expression of α-smooth muscle actin, type I collagen, transforming growth factor-β, and c-Myc in renal tissue. Western blot analysis was performed to detect protein expression levels of α-smooth muscle actin, type I collagen, transforming growth factor-β, c-Myc, Smad3, and p-Smad3 in renal tissue.
RESULTS AND CONCLUSION: (1) Through GEO-based network pharmacology analysis, nine active components of astragalus–peach kernel and seven core disease targets (c-Myc, RB1, CHUK, MAPK14, DPEP1, NR1I3, NQO2) were identified. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the Ras–MAPK–c-Myc pathway is associated with chronic kidney disease. Molecular docking suggested strong binding affinity between c-Myc and the core active compounds. (2) Compared with the blank group, the model group exhibited reduced glomerular number, structural abnormalities, pronounced inflammatory cell infiltration in renal tubules, extensive fibroblast proliferation in the renal tissue, and abnormal accumulation of collagen fibers in the renal interstitium. Serum creatinine and blood urea nitrogen levels, as well as the mRNA and protein levels of α-smooth muscle actin and type I collagen, were significantly elevated (P < 0.05). Compared with the model group, the astragalus–peach kernel group showed more intact renal histology, reduced inflammatory infiltration, and decreased fibroblast proliferation in the renal tissue. The astragalus–peach kernel treatment suppressed the elevations in serum creatinine and blood urea nitrogen levels and both mRNA and protein expression of α-smooth muscle actin and type I collagen (P < 0.05). Compared with the blank group, the model group showed significantly elevated mRNA and protein levels of c-Myc and transforming growth factor-β, as well as increased protein levels of Smad3 and p-Smad3 (P < 0.05). Compared with the model group, the astragalus–peach kernel treatment significantly downregulated mRNA and protein levels of c-Myc and transforming growth factor-β, as well as reduced protein levels of Smad3 and p-Smad3 (P < 0.05). These findings suggest that astragalus–peach kernel may slow chronic kidney disease progression in Sprague-Dawley rats, potentially through the modulation of the c-Myc/transforming growth factor-β/Smad3 signaling pathway.

Key words: astragalus, peach kernel, chronic kidney disease, renal fibrosis, c-Myc, transforming growth factor-β, α-smooth muscle actin, type I collagen

CLC Number:

Liu Jiayong, Yao Jingjing, Liu Shiyu, Tang Yi, Dong Jianing, Zhang Xin, Hou Lanwei, Kang Jianying, Zhao Yirui. Mechanism by which astragalus-peach kernel alleviates renal fibrosis in chronic kidney disease rats[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(25): 6554-6565.

Figures/Tables 6

2.1 GEO数据库结合网络药理学分析结果 2.1.1 数据集的合并及差异基因的筛选将GEO数据库中的两组数据集以|logFC|=1与P=0.05为条件获取单个数据的差异基因，并将上调和下调最显著的50个差异基因进行可视化，绘制热图和火山图(图1A-D)。使用RRA方法将2个数据集获得的差异基因进行排序，结果共得到972个差异基因，其中包含下调差异基因536个、上调差异基因436个，绘制热图(图1E)。主成分分析结果显示两组数据集有显著差异(图1F)。将数据进行归一化处理后应用Batch批次矫正法，依据|logFC|> 1及P < 0.05为阈值进行筛选，显示预处理后的合并基因表达矩阵共含有624个差异表达基因，其中上调基因数量为327个、下调基因数量为297个，绘制火山图、热图和箱线图(图1G，H)。 2.1.2 黄芪-桃仁活性成分-疾病核心靶点模型构建在TCMSP数据库中依据生物利用度及类药性条件进行筛选，得到黄芪有效成分16个、潜在靶标159个，桃仁有效成分40个、潜在靶标63个。将黄芪桃仁有效成分、靶点合并去重后共得到有效成分56种，潜在病理靶标182个，见图2。在GeneCards、Pharmgkb、TTD和DrugBank与OMIM数据库中，通过以“Chronic kidney disease”为关键词，去重整理后共得到16 282个疾病靶点(图2B)，将其与RRA和Batch法分析获得的交集差异基因(图2A)以及芪桃药对获得的潜在靶点进行合并，最终获得7个最具潜力的靶点基因(图2C)，分别为c-Myc、RB1、CHUK、MAPK14、DPEP1、NR1I3、NQO2。基于已筛选出的靶点基因，利用Cytoscape进一步构建芪桃成分-疾病网络图(图2D)，该网络包括9个药物活性成分和7个核心靶点，共有16个节点、13条边。拓扑分析提示了生物网络中双度值中位数是1，核心活性成分为槲皮素(MOL000098)。 2.1.3 GO、KEGG富集分析如图2E所示，GO通路富集分析主要包括生物过程、细胞组成、分子功能3个方面。在生物过程中，主要富集条目涉及免疫和细胞分化调控、微小RNA代谢和转录的正向调控等；细胞组成相关富集条目有RNA聚合酶Ⅱ转录调控复合体、蛋白激酶复合体、染色质沉默复合体等；分子功能的富集条目有DNA结合转录激活和抑制活性、蛋白质配体酶连接等。GO通路富集分析提示了黄芪-桃仁可能通过调节应激反应、细胞凋亡、影响转录调控复合体的功能等过程来影响慢性肾病的进展。根据KEGG富集分析结果，将最显著的30个信号通路进行可视化，结果包括MAPK信号通路、PI3K-Akt信号通路、细胞周期、细胞衰老、Ras信号通路等(图2F)。KEGG富集分析结果提示，Ras信号通路和MAPK信号通路与慢性肾脏病密切相关。Ras-MAPK信号通路可以通过提高上皮-间质转化有关转录因子(SNAIL和ZEB1)的表达[21]，驱动细胞外基质的重塑；另一方面，RAS-MAPK信号通路可以增强转化生长因子β信号传导，激活肌成纤维细胞，加剧肾纤维化进展[22]。富集在MAPK信号通路中的相关基因有c-Myc、CHUK、RASA1，这些基因功能涉及炎症、细胞周期、及细胞的的凋亡分化。结合文献发现，c-Myc不但是MAPK下游效应因子[23]，更是调控转化生长因子β活化的重要通路之一，因此作者推测黄芪-桃仁治疗慢性肾脏病可能与c-Myc有关。 2.1.4 分子对接验证在黄芪-桃仁活性成分-核心靶点的分析中共获得9种活性成分，去除未能找到分子结构的3种化合物(MOL001366、MOL001338和MOL001321)，将其余6种活性成与c-Myc进行分子对接，结果显示结合效果较好，能量均低于 0 kJ/mol。使用Pymol将结果进行可视化，见图3。 2.2 动物实验验证结果 2.2.1 实验动物数量分析 40只大鼠全部进入结果分析。 2.2.2 黄芪-桃仁改善慢性肾脏病大鼠肾功能和组织病理变化、减轻纤维化与炎症反应动物实验流程见图4A。药物治疗过程中，空白组大鼠动作灵活，好斗，皮毛光滑，饮食与尿便尚可；模型组大鼠安静少动，皮毛枯暗无华，饮食减退，体质量减轻；与模型组相比，达格列净组、黄芪-桃仁组大鼠饮食、毛发色泽、活动状态均有好转。各组大鼠体质量变化见图4B。大体观察肾脏组织外观可见，空白组肾脏组织表面光滑，呈红褐色，质地紧实；与空白组相比，模型组大鼠肾脏组织表面粗糙，凹凸不平，有纤维化触感，颜色呈苍白色，肾脏触感明显变硬；与模型组相比，达格列净组和黄芪-桃仁组肾脏组织外观均有明显改善，见图4C。苏木精-伊红染色结果显示，空白组大鼠肾脏组织形态正常，肾小球、肾小管结构清晰，无病理改变；与空白组相比，模型组大鼠肾脏组织已有明显病理改变，肾小球数量减少、结构异常，出现萎缩、硬化，肾小管有大量炎性细胞浸润，空泡样改变，肾间质大量纤维化；与模型组相比，达格列净组和黄芪-桃仁组肾脏组织形态更趋完整，空泡改变减少，肾小球萎缩情况改善，肾间质纤维化水平下降，炎性细胞浸润明显降低，见图4D。 Masson染色结果显示，空白组大鼠肾脏组织中肾小球、肾小管未见明显胶原纤维沉积，蓝色信号稀少；与空白组相比，模型组大鼠肾脏组织中出现大量成纤维细胞，肾间质中有大量胶原纤维募集；与模型组相比，达格列净组和黄芪-桃仁组大鼠肾脏组织成纤维细胞减少，肾间质中胶原纤维募集程度减轻，见图4D。为明确黄芪-桃仁对SD大鼠肾功能的影响，对肾功能相关血清指标进行了检测。结果显示，与空白组比较，模型组大鼠血清中肌酐、尿素氮水平分别升高0.8，2.3倍(P < 0.01，P < 0.001)；与模型组比较，达格列净组大鼠血清中肌酐、尿素氮水平降低(P < 0.05)，黄芪-桃仁组大鼠血清中肌酐、尿素氮水平降低(P < 0.05)，达格列净组与黄芪-桃仁组大鼠血清中肌酐、尿素氮水平比较差异无显著性意义(P > 0.05)，见图4E，F。以上结果表明，黄芪-桃仁可以改善慢性肾脏病SD大鼠一般生理状态、肾脏组织结构，降低肾功能损伤，减轻纤维化和炎症反应，在慢性肾脏病中发挥了显著治疗作用。 2.2.3 黄芪-桃仁通过抑制α-平滑肌肌动蛋白和Ⅰ型胶原表达发挥抗纤维化作用为验证黄芪-桃仁对肾纤维化的疗效，此次实验对肾纤维化的关键指标α-平滑肌肌动蛋白、Ⅰ型胶原进行验证。免疫组化染色结果显示，与空白组相比，模型组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原表达明显增加，差异有显著性意义；与模型组相比，达格列净组与黄芪-桃仁组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原表达均减少，差异有显著性意义；达格列净组与黄芪-桃仁组大鼠肾脏组织中α-平"

滑肌肌动蛋白、Ⅰ型胶原表达比较差异无显著性意义(P > 0.05)，见图5A-C。 RT-qRCR 检测结果显示，与空白组相比，模型组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原mRNA表达升高(P < 0.01，P < 0.001)；与模型组相比，达格列净组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原mRNA表达降低(P < 0.01)，黄芪-桃仁组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原mRNA表达降低(P < 0.01)；达格列净组与黄芪-桃仁组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原mRNA表达比较差异无显著性意义(P > 0.05)，见图5D，E。 Western blot检测结果显示，与空白组相比，模型组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原蛋白表达升高(P < 0.001，P < 0.01)；与模型组相比，达格列净组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原蛋白表达降低(P < 0.01，P < 0.05)，黄芪-桃仁组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原蛋白表达降低(P < 0.01，P < 0.05)，达格列净组与黄芪-桃仁组大鼠肾脏组织中α-平滑肌肌动蛋白、Ⅰ型胶原蛋白表达比较差异无显著性意义(P > 0.05)，见图5F-H。以上结果说明，黄芪-桃仁可能通过抑制α-平滑肌肌动蛋白、Ⅰ型胶原的表达发挥了抗纤维化作用。 2.2.4 黄芪-桃仁通过抑制c-Myc和转化生长因子β/Smad3信号通路减轻肾组织纤维化为证实GEO数据库结合网络药理学分析预测的关键靶点c-Myc在慢性肾脏病中的作用，此次实验对c-Myc及其相关通路的基因与蛋白表达进行了检测。RT-qRCR与Western blot检测结果显示，模型组大鼠肾脏组织中c-Myc、转化生长因子β的mRNA和蛋白表达均高于空白组，差异均有显著性意义，其中c-Myc、转化生长因子β mRNA表达分别上调约1倍和24.88倍，c-Myc、转化生长因子β蛋白表达分别增加约1.3倍和1.8倍；与模型组相比，达格列净组和黄芪-桃仁组肾脏组织中c-Myc、转化生长因子β的mRNA和蛋白表达均降低，差异均有显著性意义(P < 0.05)，见图6。 RT-qRCR与Western blot检测结果显示，与空白组相比较，模型组大鼠肾脏组织中Smad3、p-Smad3蛋白表达分别上调约0.9倍和1.2倍，差异有显著性意义；与模型组比较，达格列净组与黄芪-桃仁组大鼠肾脏组织中Smad3、p-Smad3蛋白表达均降低，差异有显著性意义；达格列净组与黄芪-桃仁组大鼠肾脏组织c-Myc、转化生长因子β的mRNA与蛋白表达以及Smad3、p-Smad3蛋白表达比较差异均无显著性意义(P > 0.05)，见图6。以上结果说明，黄芪-桃仁可以通过抑制c-Myc信号通路下调转化生长因子β/Smad3的表达，减轻肾组织的纤维化程度，从而发挥治疗慢性肾脏病的作用。"

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