The pathophysiology of DFUs is relatively well studied, and growing evidence points towards the regulation of a key player involved in wound healing. The dysregulated macrophage responses are associated with impaired healing of diabetic wounds and are therefore considered a therapeutic target to hasten wound healing (Huang et al., 2021). It has been well established that the transcription factor Nuclear factor erythroid-2 related factor (Nrf2) suppresses macrophage inflammatory responses by blocking pro-inflammatory cytokine production (Kobayashi et al., 2016). The role of Nrf2 in modulating the inflammatory response during tissue damage-induced inflammation (Eichenfield et al., 2016), including the production of pro-inflammatory cytokines IL-6, IL-8, and TNF-α among others (Chen et al., 2021) and the modulation of this transcription factor, such as through exogenous activation (Shao et al., 2019), thereby dampening inflammation in macrophages (Lin et al., 2022), provides a formidable means of therapy towards promoting wound healing. However, the complex molecular machinery involved in Nrf2-mediated regulation in macrophages and its modulation remains to be delineated.

Mouse skin excisional wounds are reliable models for studying human wounds, including the necessary complexities that capture healing responses (Chen et al., 2015a). The apparent lack of a reliable in vitro model to study macrophage responses in experimental hyperglycemia enabled us to develop such models as in (Ganesh and Ramkumar, 2021) and (Ganesh and Ramkumar, 2023) with the use of IC-21 macrophage cell line derived from C57BL/6 mice (Chamberlain et al., 2009) that display characteristic M2 markers such as CD206 with relevance to wound healing (Shook et al., 2016). In addition, assessing macrophage responses in wound tissue environment typically involves studying macrophage-fibroblast interaction and the mouse L929 fibroblast cells that are well documented towards testing cell proliferation, migration, and other relevant experimentation (Sung et al., 2020), and, in the development of wound dressings (Srivastava et al., 1993). Interestingly, conditioned media from L929 cells serve as a source of M-CSF that is used routinely to differentiate bone marrow-derived macrophages isolated from the bone marrow (Heap et al., 2021). Concerning the in vivo model, streptozotocin (STZ)-induced male C57BL/6 mice have been proven as a suitable diabetic wound model (Saadane et al., 2020). While it is well documented that STZ-induced diabetic mice suffer from an impairment of wound closure in comparison to untreated mice, wound closure was further delayed in STZ-induced diabetic NRF2 knock-out mice (Long et al., 2016), suggesting the crucial role of Nrf2 in facilitating wound closure in pathological situations such as diabetes. Several reports highlight that the activation of Nrf2 can inhibit the M1 polarization and promote the M2 polarization through multiple signaling pathways, including TGF-β/SMAD, TLR/NF-κB, and JAK/STAT pathways (Wang and He, 2022). On the other hand, issues regarding the involvement of Nrf2 (Joshi and Werner, 2017) and M2 polarization of macrophages in hastening wound healing, such as the findings of Jetten et al. (2014), have raised intrigue in the molecular pathophysiology of DFU and its healing.

We reported previously that hyperglycemia exposure to macrophages suppresses Nrf2 activation, resulting in lowered expression of Nrf2 downstream mediator HO1 and the macrophage mannose receptor CD206 and its amelioration by pterostilbene or PTS (Ganesh and Ramkumar, 2021). PTS is a structural analog of resveratrol but with a more favorable pharmacokinetic profile (Qiu-YiChoo et al., 2014) and has already been evaluated for treating burn injuries (Xie et al., 2021). We have reported PTS for its varied beneficial effects, including anti-apoptotic effects in pancreatic beta cells (Bhakkiyalakshmi et al., 2014) and in diabetic mice (Sireesh et al., 2017) and lowering macrophage dysregulation induced by hyperglycemia (Ganesh and Ramkumar, 2021) through the regulation of Nrf2. One of our recent studies also demonstrated PTS-mediated effects in macrophage M2 polarization and reducing inflammation (Ganesh and Ramkumar, 2023) and the beneficial role of the compound is also reported in other studies through down-regulation of macrophage M1 polarization (Pan et al., 2008) by hindering NF-κB activation and blunting oxidative stress in topical wound therapy in mice (Hu et al., 2022). We also utilized the commercially available recombinant platelet-derived growth factor, Regranex (Papanas and Maltezos, 2008), to treat DFUs as a reference standard to assess PTS-mediated regulation of wound healing responses.

In the present study, we investigate the potential of PTS in promoting the wound healing response in diabetic mice via regulation of Nrf2 that, in turn, modulates the inflammatory wound environment, and, in vitro, to assess the effect of macrophage conditioned media, obtained from such cells exposed to experimental hyperglycemia with or without PTS intervention, in fibroblast response to wounding to attempt emulate diabetic wound tissue wherein macrophages modulate wound fibroblasts under different cellular contexts.