Even in physically healthy people, burn injuries are frequently accompanied by consequences. People with diabetes may have a higher risk of complications and death. Wound healing failures and infections are also common in people with diabetes.[22]Because numerous components of wound healing physiology are disrupted in diabetes wounds, resulting in delayed wound healing and persistent inflammation. Less endothelial progenitor cells, lower endothelium-derived nitric oxide synthase activity, a growth factor shortage, reduced macrophage activity, reduced collagen deposition, increased ECM proteolysis, and the switch from M1 to M2 phenotype are all examples of molecular imbalances.[23]For resolving inflammation and changing the balance toward tissue repair, this transition from M1 to M2 is critical.[24]
Ginsenoside Rb1 (0.5% w/w) has been reported to have a potent healing effect on burn wounds by several mechanisms, including enhanced vascularization in the surrounding tissue, production of Interleukin 1 beta (IL-1β) and vascular endothelial growth factor (VEGF) from the burn wound. The stimulation of VEGF synthesis and increases in expression of hypoxia-inducible factor (HIF)-1 in kerationcytes and an increase in IL-1 owing to macrophage buildup in the burn site are all contribute to angiogenesis. Also, by promoting the bio-active substances (histamine, SP, and MCP-1),ginsenoside Rb1(0.5% w/w) facilitate burn wound healing.[25]
Ginsenosides was also reported to promote wound healing by activating the mitogen-activated protein kinase pathway, stimulating intracellular cAMP levels and associated protein expression in the nucleus, enhancing the dermal fibroblast proliferation and collagen synthesis. Furthermore, ginsenoside Rb1 enhances skin keratinocyte movement and myofibroblast transformation in senescent dermal fibroblasts of human skin by stimulating the production of growth factors, including a sequence of SASP factors. [26]In addition to the above mechanism,M1 to M2 transition is crucial, as it shifts the wound from the inflammatory phase to tissue healing.
Wound healing is a complicated biological process divided into four stages: haemostasis phase (0–several hours after damage), inflammation phase (1–3 days), proliferation phase (4–21 days), and remodelling phase (21 days–1 year). [27] Any of these interrupted stages leads to poor healing, such as chronically difficult-to-heal ulcers or extensive scarring, which has a significant and rising health and cost burden on our society.[27–29]The transition from the inflammatory stage to the regenerative stage of wound healing is vital, and evidence is growing that a faulty transition is associated to wound healing difficulties. As a result, therapeutic developments focussing on this shift could be justified.[18]In order to protect from infections and removing dead tissues, the inflammatory phase is necessary as it brings haemostasis and activates innate immune system.[30]On the other hand, if the inflammation is prolonged, it may interfere with keratinocyte differentiation and activation, and obstruct wound healing from progressing through the usual stages.[28] Furthermore, persistent inflammation in chronic inflammatory situations, such as diabetic wounds, is expected to raise metalloproteinases and other proteases, which degrade ECM components and growth factors essential for healing.[23]Furthermore, a lot of scarring has been associated with persistent inflammation.[31]
During wound healing, macrophages switch from a pro-inflammatory M1 phenotype to a tissue-repair M2 phenotype. This produces anti-inflammatory mediators like decoy IL-1 receptor type II, IL-10, and IL-1R antagonist, as well as bioactive molecules like VEGF, IGF1 and TGF that promote ECM synthesis, fibroblast proliferation, and angiogenesis.[32, 33]The transition from M1 to M2 is crucial for resolving inflammation and shifting the balance toward tissue healing.[24]In both animal and human wounds, continuous IL-1 β (pro-inflammatory cytokines) blocked the upregulation of proliferator-activated receptor (PPAR)γ activity, which is essential for macrophage phenotypic transformation. As a result, it was discovered that diabetes induces a faulty M1–M2 transition, which delays wound healing.[34]As a result, regulation of the above pathways is required for optimal wound healing.
In addition to the burn wound repair investigation, we included two additional experimental groups (Groups I to V) to investigate the differences in CD68 (M1 phenotype) and CD163 protein expression (M2 phenotype). On day 5, the current study found that ginsenoside Rb1 (0.5% w/w) had elevated CD163 (2.1 ± 0.12) and lowered CD68 (1.2 ± 0.09), compared to diabetic control ratsthat had decreased CD163 (0.98 ± 0.07) and increased CD68 (2.6 ± 0.18)(Table-4). CD68 and CD163 are glycoproteins and markers of wound healing macrophages. This transition from M1 to M2 phenotype is crucial in diabetic wounds, and the findings highlight the mechanism behind enhanced wound healing of ginsenoside Rb1(0.5% w/w) in diabetic animals with burn wounds.Furthermore, the reduced concentrations of TNF-α and IL-1β on day 5 in the ginsenoside Rb1(0.5% w/w) group (Figure-3) further supports the transition from M1 to M2 phenotype. The low TNF-α and IL-1β level are sustained throughout the healing period in ginsenoside Rb1groups (Figure-3).
Further, the current research findings haverecorded erythema, thickness, reduced collagen and inflammation in control group animals (Fig. 1&2), which were practically recovered to normal in ginsenoside Rb1 (0.5% w/w)treated groups, with maximal burn wound closure (99.23 ± 3.41) suggesting considerable (P < 0.05) burn wound healing activity. This might be related to ginsenoside Rb1 anti-inflammatory, antioxidant, and cell growth-promoting properties.[35]Improved tensile strength may be aided by collagen production, angiogenesis, maturation, and fibre stability.[36] The levels of hydroxyproline and hexosamine in the tissue were examined since they are directly related to collagen production and extracellular matrix development, respectively.[37] Whenginsenoside Rb1(0.5% w/w) treated burn wounds were compared to untreated diabetic control rats, significantly higher levels of hydroxyproline and hexosamine (Table-2) were found (P < 0.05). Ginsenoside Rb1 activates macrophages, releasing cytokines and growth factors with antibacterial and anti-inflammatory properties and promotingmigration of dermal fibroblast to the lesion. In the wound, these fibroblasts multiply, creating extracellular matrix (ECM) biomaterials such as collagen to start the healing process.[38–40]
The pro-inflammatory mediator IL-6 (Table-3) was noticed as soon as 12–24 hours after cutaneous damage, and these ingredients promote angiogenesis, which is essential in the inflammatory stage of wound healing.[41] More intriguingly, the outcomes of this investigation revealed that ginsenoside Rb1(0.5% w/w) did not affect IL-6 levels on day two samples (Table-3). This shows that during the early phases of recovery, ginsenoside Rb1(0.5% w/w) did not affect pro-inflammatory cytokines produced by macrophages. On the other hand, Ginsenoside Rb1(0.5% w/w) therapy increased IL-10 levels on day ten following burn injury. It's worth mentioning that IL-10 is a cytokine generated by T cells and macrophages with anti-inflammatory characteristics.42The wound-healing environment appears to be altered by IL-10, which seems to reduce the expression of profibrotic/proinflammatory mediators, leading to a reduction in inflammatory cell recruitment to the wound.[42, 43] Treatment with ginsenoside Rb1(0.5% w/w) increased serum IL-10 levels while decreasing IL-6 expression, especially on day ten after burn injury. As a result, ginsenoside Rb1 regulates proinflammatory and anti-inflammatory cytokines and the systemic immunological pathways that relate them to cellular proliferation.
Biochemical analysis of plasma samples was performed to determine the function of anti-oxidants, pro-inflammatory, and anti-inflammatory mediators behind the beneficial effect of ginsenoside Rb1. In our research, ginsenoside Rb1(0.5% w/w) shown extraordinary antioxidant activity by substantially (P < 0.05) boosting the levels of antioxidant enzymes like SOD, CAT, and glutathione (GSH), suggesting that ginsenoside Rb1 could aid in the prevention of oxidative damage and the improvement of the healing process (Table-2).
SOD-1 catalyzes the dismutation of superoxide radicals into dioxygen and hydrogen peroxide (H2O2), which are both potentially hazardous. The CAT activity of the ginsenoside Rb1(0.5% w/w) treated group was much higher, suggesting that elevated CAT may effectively neutralize H2O2 accumulated due to enhanced SOD activity. [44–46] GSH is also a critical endogenous thiol antioxidant that acts as a supporting factor for glutathione peroxidase (GPx) in removing lipid hydroperoxide.[46]Furthermore, when reactive oxygen species destroy polyunsaturated lipids, MDA, a secondary metabolite of LPO,is utilized to determine the level of osmotic damage in an organism.[47]In this study, ginsenoside Rb1(0.5% w/w) significantly lowered blood MDA levels (7·32 ± 1·51) when compared to diabetic controls (12·23 ± 1·03). Notably, the diabetic control animals in this study had lower anti-oxidant levels and greater MDA levels, which might explain why their burn wounds took longer to heal.