Danlian-Tongmai formula improves diabetic vascular calcification by regulating CCN3/NOTCH signal axis to inhibit inflammatory reaction

Abstract

Background: Vascular calcification (VC) commonly occurs in diabetes and is associated with cardiovascular disease incidence and mortality. Currently, there is no drug treatment for VC. The Danlian-Tongmai formula (DLTM) is a traditional Chinese medicine (TCM) prescription used for diabetic VC (DVC), but its mechanisms of action remain unclear. This study aims to elucidate the effects of DLTM on DVC and explore the underlying mechanisms of action.

Methods: Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to identify the metabolites of DLTM. A DVC rat model was established using streptozotocin (STZ) combined with vitamin D3 (VitD3). The effects of DLTM on DVC were evaluated through alizarin red staining, calcium deposition, and changes in osteogenic and contractile markers. The specific molecular mechanism of DLTM in treating diabetic VC was comprehensively analyzed by transcriptomics, molecular docking and in vivo experimental verification.

Results: We identified 108 major metabolites of DLTM. In vivo, high-dose DLTM significantly alleviated VC in diabetic rats. Transcriptomic analysis showed that DLTM treatment markedly altered the transcriptomic profile of rat aortas, which was associated with regulating the CCN3/NOTCH signaling pathway, promoting vascular smooth muscle contraction, and inhibiting the inflammatory responses. Molecular docking and molecular dynamics simulation demonstrated strong binding interactions between DLTM metabolites and key molecules within the CCN3/NOTCH pathway, including NOTCH1, DLL1, DLL4, hes1, and hey1. In vivo experiments confirmed that DLTM could upregulate CCN3, inhibit the activation of NOTCH signaling ligands DLL1 and downstream transcription factors hes1 and hey1, and reduce the release of inflammatory cytokines IL6, IL1β, and TNFα.

Conclusion: DLTM alleviates DVC by regulating the CCN3/NOTCH signaling axis to inhibit inflammatory responses. Our research provides experimental basis for clinical treatment and drug transformation of diabetic VC.

Keywords: CCN3; Danlian-Tongmai formula; UHPLC-MS; notch signaling; vascular calcification.

FIGURE 1

The chromatographic fingerprint of DLTM. (A) Ion flow diagram of DLTM in negative ion mode. (B) Ion flow diagram of DLTM in positive ion mode. Molecular weight and formula of these metabolites are listed in Supplementary Table S2.

FIGURE 2

Ameliorative effect of DLTM on vascular calcification in DVC rat. (A) Schematic diagram of the experimental design for the rat model; (B) Body weight of rats after receiving VitD₃+STZ or vehicle injection and oral administration of different doses of DLTM, Dapagliflozin, or vehicle for 4 weeks (n = 7–11); (C) Fasting blood glucose levels of rats after receiving VitD₃+STZ or vehicle injection and oral administration of different doses of DLTM, Dapagliflozin, or vehicle for 4 weeks (n = 7–11); (D) Serum calcium levels in each group of rats (n = 5); (E) Serum phosphorus levels in each group of rats (n = 5); (F) Representative images of whole aorta alizarin red staining in each group of rats (n = 3); (G) Vascular ALP levels of each group of rats (n = 5); (H). Vascular calcium content of each group of rats (n = 5); (I) Representative images of alizarin red-stained aortic arch sections in each group of rats (n = 3,4 × 20×); (J) Quantitative analysis of the alizarin red positive staining area in aortic sections from I (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 3

DLTM alleviated osteogenic differentiation of VSMC in vivo. (A) Representative Western blot images of RUNX2, BMP2, αSMA, and SM22α expression in the aortae of rats from each group; (B) Quantitative analysis of RUNX2 expression in (A, C) Quantitative analysis of BMP2 expression in (A, D) Quantitative analysis of αSMA expression in (A, E) Quantitative analysis of SM22α expression in (A). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

FIGURE 4

Transcriptome sequencing reveals the mechanism of DLTM against diabetic VC. (A) Volcano plot of transcriptome sequencing showing DEGs between the DVC group and CON group, with thresholds of P < 0.05 and |log2 (Fold Change) | ≥ 1.2; (B) Volcano plot of transcriptome sequencing showing DEGs between the DLTM group and DVC group, with thresholds of P < 0.05 and |log2 (Fold Change) | ≥ 1.2; (C) Venn diagram showing DEGs between DVC and CON groups, DLTM and DVC groups, with enrichment analysis and PPI analysis of overlapping DEGs to identify core DEGs; (D) GO enrichment analysis results of overlapping DEGs; (E) KEGG enrichment analysis results of overlapping DEGs; (F) GSEA analysis results of transcripts; (G). Heatmap of Top10 core DEGs, (H) The mRNA expression levels of calcium-related hubDEGs among the CON, DVC, and DLTM groups (n=3). N: CON group, M: DVC group, D: DLTM group. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

FIGURE 5

Metabolites of DLTM exhibited good binding affinities to molecules in CCN3/NOTCH signaling pathway. (A) Heatmap of binding energies between potentially effective metabolites of DLTM and molecules in CCN3 and NOTCH signaling pathways (see Supplementary Table S6 for details); (B–G) Molecular docking images showing the DLTM metabolites with the highest binding energies to CCN3 and various NOTCH signaling molecules, (B) CCN3- Worenine (−8.9 kcal/mol); (C) NOTCH1- Tormentic acid (−8.2 kcal/mol); (D) hey1- Tormentic acid (−7.2 kcal/mol); (E) hes1 (−8.6 kcal/mol)- Worenine; (F) DLL1-Tormentic acid-(−7.9 kcal/mol); (G) DLL4-Worenine (−7.7 kcal/mol).

FIGURE 6

DLTM inhibits the release of inflammatory factors by regulating CCN3/NOTCH signal axis. (A) Representative Western blot images of CCN3, DLL1, DLL4, NOTCH1, hey1, hes1 expression in the aortas of rats from the CON, DVC, and DLTM groups; (B) Quantitative analysis of CCN3, DLL1, DLL4, NOTCH1, hey1, hes1 expression (n = 3); (C) Serum TNFα levels in rats from the CON, DVC, and DLTM groups (n = 5); (D) Serum IL1β levels in rats from the CON, DVC, and DLTM groups (n = 5); (E) Serum IL6 levels in rats from the CON, DVC, and DLTM groups (n = 5). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

FIGURE 7

The mechanism diagram of DLTM against diabetic VC.