Asiaticoside-nitric oxide promoting diabetic wound healing through the miRNA-21-5p/TGF-β1/SMAD7/TIMP3 signaling pathway

Centella asiatica (L.) Urban is an ethnobotanical herb. The main bioactive components of Centella asiatica are pentacyclic triterpenoid glycosides, namely asiaticoside and hydroxyasiaticoside. Asiaticoside possess a diverse array of pharmacological properties, such as wound-healing, anti-inflammatory, antioxidant, anti-allergic, antidepressant, anxiolytic, anti-fibrotic, antibacterial, anti-arthritic, anti-tumor, and immunomodulatory activities. The purpose of this investigation is to explore potential therapeutic interventions for the delayed healing of wounds in diabetic patients (DW) facilitated by Asiaticoside-Nitric Oxide. To clarify the key molecular mechanism of miRNA-21-5p in DW wound repair and to deepen the understanding of DW disease pathogenesis. Firstly, miRNA microarray technology, bioinformatics, and RT-qPCR were used to analyze DW patients' and normal controls' skin tissue samples. Secondly, in order to investigate the role of miRNA-21-5p, a hyperglycemic model was established using HaCaT cells. Overexpressing as well as interfering HaCaT cell lines were constructed by lentiviral infection to further explore the proliferative and migratory effects of Asiaticoside-Nitric Oxide. The next step was to search for potential target genes of miRNA-21-5p and verify them with dual-luciferase reporter assay. Finally, the expression levels of target genes and proteins were detected through the utilization of RT-qPCR and Western blotting under the influence of Asiaticoside-Nitric Oxide. A library of miRNAs and target genes expressed explicitly in DW patients and rats was established. The study confirmed the upregulation of miRNA-21-5p in DW patients and identified its involvement in signaling pathways related to chronic ulcer wound repair. Overexpression of LV-miRNA-21-5p significantly promoted cell proliferation, while treatments of Asiaticoside-Low dose (AC-L) and Asiaticoside-Medium dose (AC-M) enhanced proliferation and migration, particularly when combined with nitroprusside (SNP). Further analysis revealed potential target genes of miRNA-21-5p, such as TGF-β1, SMAD7, and TIMP3. Their interaction with miRNA-21-5p was confirmed through dual luciferase assays. The study found that anti-DW drugs increased the expression of TGF-β1 and SMAD7 while inhibiting TIMP3 expression in a high-glucose environment. The research concluded that miRNA-21-5p plays a crucial role in the delayed healing of diabetic wounds, and that the combination treatment of AC + SNP shows promise in promoting wound healing in DW rats. Target genes, including TGF-β1, SMAD7, and TIMP3, may contribute to the regulatory mechanisms involved in diabetic wound healing. These findings provide valuable insights for developing novel therapeutic approaches for DW.