College of Pharmaceutical Sciences, Translational Pharmacy Center of Medical Research Institute, Southwest University, Chongqing, 400716, China.
Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, School of Life Sciences, Southwest University, Chongqing, 400715, China.
J Ethnopharmacol. 2021 Apr 24;270:113806. doi: 10.1016/j.jep.2021.113806. Epub 2021 Jan 12.
Rhizoma Coptidis (RC) is a traditional Chinese medicine (TCM) used for treating diabetes (Xiao Ke Zheng), which is firstly recorded in Shennong Bencao Jing. Modern pharmacological studies have confirmed that RC has beneficial effects on diabetes and its complications. Alkaloids are the main active pharmacological component of RC. However, the effect and molecular mechanism of total Rhizoma Coptidis alkaloids (TRCA) in improving diabetic nephropathy (DN) are still unclear.
To verify the effect of TRCA in the treatment of DN and clarify the molecular mechanism by combining network pharmacology and transcriptomic.
Eight-week-old db/db mice were orally administered with normal saline, 100 mg/kg TRCA, and 100 mg/kg berberine (BBR) for 8 weeks. Serum, urine, and kidney samples were collected to measure biological indicators and observe renal pathological changes. Then, the molecular mechanism of TRCA improving DN was predicted by the network pharmacology. Briefly, the main active alkaloids components of TRCA and their targets were collected from the database, as well as the potential targets of DN. Using the Cytoscape software to visualize the interactive network diagram of "ingredient-target". The GO and KEGG pathways enrichment analysis of the core targets were executed by Metascape. Furthermore, RNA-seq was used to get whole transcriptomes from the kidneys of db/m mice, db/db mice, and db/db mice treated with TRCA. The key differentially expressed genes (DEGs) were gathered to conduct the GO and KEGG pathways enrichment analysis. Finally, the potential pathways were validated by western blotting.
The administration of BBR or TRCA for 8 weeks significantly reduced the fasting blood glucose (FBG) and body weight of db/db mice, and improved their renal function and lipid disorders. According to H&E, PAS, and Masson staining, both the BBR and TRCA could alleviate renal damage and fibrosis. The Venn diagram had shown that seven alkaloids ingredients collected from TRCA regulated 85 common targets merged in the TRCA and DN. The results of RNA-seq indicated that there are 121 potential targets for TRCA treatment on DN. Intriguingly, both the AGE-RAGE signaling pathway and the PI3k-Akt signaling pathway were included in the KEGG pathways enrichment results of network pharmacology and RNA-seq. Moreover, we verified that TRCA down-regulated the expression of related proteins in the AGEs-RAGE-TGFβ/Smad2 and PI3K-Akt pathways in the kidney tissues.
In summary, the renal protection of TRCA on DN may be related to activation of the AGEs-RAGE-TGFβ/Smad2 and PI3K-Akt signaling pathways.
黄连（RC）是一种传统的中药，用于治疗糖尿病（消渴症），首次记录在《神农本草经》中。现代药理学研究证实，RC 对糖尿病及其并发症有有益的作用。生物碱是 RC 的主要活性药理成分。然而，总黄连生物碱（TRCA）改善糖尿病肾病（DN）的效果和分子机制仍不清楚。
通过网络药理学和转录组学相结合，验证 TRCA 治疗 DN 的效果，并阐明其分子机制。
8 周龄 db/db 小鼠连续 8 周给予生理盐水、100mg/kg TRCA 和 100mg/kg 黄连素（BBR）灌胃。收集血清、尿液和肾脏样本，以测定生物指标并观察肾脏病理变化。然后，通过网络药理学预测 TRCA 改善 DN 的分子机制。简而言之，从数据库中收集 TRCA 的主要活性生物碱成分及其靶点，以及 DN 的潜在靶点。使用 Cytoscape 软件可视化“成分-靶点”的互作网络图。使用 Metascape 对核心靶点进行 GO 和 KEGG 通路富集分析。此外，使用 RNA-seq 从 db/m 小鼠、db/db 小鼠和 TRCA 处理的 db/db 小鼠的肾脏中获得全转录组。收集关键差异表达基因（DEGs）进行 GO 和 KEGG 通路富集分析。最后，通过 Western blot 验证潜在通路。
连续 8 周给予 BBR 或 TRCA 可显著降低 db/db 小鼠的空腹血糖（FBG）和体重，改善其肾功能和脂质代谢紊乱。根据 H&E、PAS 和 Masson 染色，BBR 和 TRCA 均可减轻肾脏损伤和纤维化。Venn 图显示，从 TRCA 中提取的七种生物碱成分调节了网络药理学和 RNA-seq 中合并的 85 个共同靶点。RNA-seq 的结果表明，TRCA 治疗 DN 有 121 个潜在靶点。有趣的是，AGE-RAGE 信号通路和 PI3k-Akt 信号通路均包含在网络药理学和 RNA-seq 的 KEGG 通路富集结果中。此外，我们验证了 TRCA 下调了肾脏组织中 AGEs-RAGE-TGFβ/Smad2 和 PI3k-Akt 通路相关蛋白的表达。
综上所述，TRCA 对 DN 的肾脏保护作用可能与激活 AGEs-RAGE-TGFβ/Smad2 和 PI3k-Akt 信号通路有关。
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