The in vivo and in vitro Investigations of Astragali Radix and Rehmanniae Radix Formula in Diabetic Wound Healing and Its Mechanisms of Actions

Diabetic foot ulcer is ulceration in the lower extremity due to neuropathy and vasculopathy, imposing risk of limb amputation in diabetic patients. In our previous clinical studies, two Traditional Chinese medicine (TCM) formulae 1 and 2 prevented 85% diabetic foot ulcer patients from limb amputation. A modified Chinese 2-herb formula (NF3) with Astragali Radix (AR) and Rehmanniae Radix (RR) in the ratio of 2:1 was established for investigation of the diabetic wound healing efficacies and its underlying mechanisms of actions.;We employed the in vivo streptozotocin (STZ)-induced diabetic foot ulcer rat model and various in vitro cell models including human skin fibroblasts (Hs27), human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMEC-1), rat bone marrow (BM)-derived endothelial progenitor cells (EPCs) and murine monocyte/macrophage RAW264.7 cells (RAW264.7 cells), for studying the respective proliferation, neovascularization (angiogenesis and vasculogenesis) and anti-inflammation potential of NF3.;We firstly demonstrated that NF3 could significantly stimulate the wound closure of diabetic foot ulcer rats through regulation of tissue epithelization of Hs27 fibroblasts, angiogenesis of HUVEC and HMEC-1, and anti-inflammation of macrophage RAW264.7 cells. Since peripheral vascular defects were contributed to the pathophysiology of diabetic foot ulcer, we further illustrated that NF3 could significantly promote the wound healing of diabetic foot ulcer rats with hindlimb ischemia through enhancement of circulating EPCs for vasculogenesis, blood capillary formation, up-regulation of VEGF and eNOS expression, and increase in MMPs activities in the ischemic muscles associated with the reduction of oxidative stress. These findings provided scientific evidences towards the diabetic wound healing efficacies of NF3.;The study focused on the effects of NF3 on neovascularization using different functional and mechanistic approaches. AR is the principal herb in NF3 and the synergistic interaction of AR and RR in NF3 exerted significant pro-angiogenic effects on HUVEC and HMEC-1. Identification of the global genomic and proteomic targets in NF3-treated HUVEC was performed to explore the signaling pathways regulated by NF3 in wound healing angiogenesis. The microarray results revealed that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The target genes were involved in transcriptional and translational regulation, cell proliferation, cell migration, tubular formation, angiogenesis, anti-inflammation, anti-oxidation and anti-tumorogenesis. Meanwhile, the proteomic results demonstrated that different differentially expressed proteins were responsible for the overall wound healing angiogenesis process in NF3-treated HUVEC.;EPCs were highly devoted to vasculogenesis in wound tissues by de novo synthesis of blood vessels. Our results demonstrated that NF3 could significantly increase circulating EPCs in diabetic foot ulcer rats through restoration of chemokine stromal derived factor-1alpha, promoting the recruitment and homing of EPCs to wound area. BM-derived EPCs were successfully isolated and were shown to possess significant proliferation, adhesion, migration and tubular formation ability in vitro after NF3 treatment.;In conclusion, our study highlighted the wide range of beneficial effects of NF3 on neovascularization and wound healing perspectives. We provided concrete scientific evidences in support of the development of NF3 into an alternative TCM therapy for the management of diabetic foot ulcer.