When used in burns or trauma, AgNPs can effectively promote wound healing, not only because of their bacteriostatic effect but also their inhibition of inflammation. This indicates that AgNPs can improve healing of the wound partly based on their antibacterial effect. In vivo and in vitro studies have shown that the mechanism by which AgNPs promote wound repair should be their anti-inflammatory effect. Inflammation and microbial growth are two important factors affecting wound healing. In most cases, the two factors arise concurrently and affect each other. Wound infection inevitably cause inflammation. Continuous inflammation and repeated infections are associated with progressive necrosis of burns wound.
The normal wound healing process includes a bleeding phase, inflammation phase, repair phase, and reconstruction phase. This seems to go along with the zoning of the wound, namely, the coagulation necrosis zone, the stasis zone, and the hyperemia zone. The hyperemia zone is the marginal area of a burn wound, with the most robust chemotactic accumulation of inflammatory cells, and the damage in this zone is reversible. In the inflammatory response process, in which many monocytes chemotactically aggregate at a site, macrophages play an irreplaceable role because they can engulf necrotic cells and infected microorganisms and can also secrete many chemokines and cytokines to regulate collagen secretion, cell proliferation, growth, and vascularization granulation [16, 17]. The two major pathways of macrophage activation include classical activation pathway (M1) and selective activation pathway (M2) [18, 19]. Scientists have studied the gene expression of the two types of activated macrophages at a wound using gene chip technology, and they found that 11 M1 marker genes and seven M2 marker genes were expressed 2 days after a burn. However, between 4 and 8 days after burn, the gene expression shifted to one M1 gene and nine M2 genes expressed. This indicates that the number of each type of activated macrophage may change during the wound healing process. In the middle and late stages of healing, M2 macrophages predominate [20]. These findings show that different types of macrophage activation during the wound healing process fluctuate in an orderly way. When the regular macrophage activation process is disrupted, normal wound healing may stagnate. This can lead to repeated vicious cycles of inflammation and infection.
The hyperemia and stasis zones in the burn wound are in the edge of the wound but the center of the inflammatory storm. How to effectively and correctly induce the inflammatory response in these zones for wound healing is the core of this study. In this study, we employed AgNPs to treat burn wounds and investigated their regulation of the inflammatory response in the region, in an attempt to reduce the occurrence of progressive necrosis of the wound. The mechanism is related to the activation of macrophages in the wound: AgNPs can specifically activate M2 macrophages to effectively reduce progressive necrosis at the wound margin.
The mechanism by which they promote healing has always been attributed wholly to their effective bacteriostatic effect. In this study, we hypothesized that the mechanism by which they effectively improve wound inflammation and relieve progressive necrosis of the wound is related to activation of macrophages. The AgNPs in the wound may exert their antibacterial effects while being engulfed by macrophages to affect the activation of these cells. Therefore, we hypothesize that AgNPs can affect the activation of M2 macrophages within a specific time frame to repair and rebuild damaged tissue.
Through proteomics research, scientists have discovered that AgNPs can inhibit the expression of pro-inflammatory factors. The mechanism may be due to the antimicrobial activity of AgNPs and their effect on MMP activity [13, 21]. AgNPs can inhibit the secretion of M1-type macrophage-related factors TNF-α and iNOS [22, 23]. After AgNPs enter the circulatory system, they can be engulfed by blood cells to cause the apoptosis of blood cells and lower the expression of TNF-α and IL-5 [24, 25]. In contrast, the expression of TGF-β1 is very low in the nonscarring healing site of fetal skin. Indeed, TGF-β antagonism can significantly reduce inflammation in healed wounds and scars compared with control treatments. All these data indicate that TGF-β plays a vital role in tissue fibrosis and scar formation [26–27].
M2 macrophage-related protein Arginase-1, MHC-II, and FIZZ-1, play an important role in wound tissue reconstruction. It reduces collagen shrinkage by secreting collagenase to reduce collagen synthesis and deposition [28]. They can reduce the infiltration of mononuclear cells and M1 macrophages in the wound, inhibit the secretion of pro-inflammatory factors, and effectively regulate the inflammation of the wound [29–30].
The activation of macrophages and the occurrence and development of inflammation have a profound effect on the wound healing and. Under normal circumstances, a reasonable and orderly inflammatory response can ensure normal and rapid healing. In this study, immunohistochemistry, RT-PCR, and Western blot were used to study the expression of inflammatory factors. A reliable surface marker of macrophages, CD68 can be expressed in cells such as macrophages, monocytes, and Kupffer cells and is a direct indicator of inflammation. M1 macrophage-associated TNF-α is potential promoter of fibroblast proliferation and are usually expressed low in fetal wounds [25]. Overexpression of these factors at a wound is associated with delayed wound healing. The low expression of TGF-β, CD68, and IL-6, which indicated downregulation of the M1 macrophage response, is the basis for the improvement of inflammation and can be used as an index to predict wound healing and scarring. Western blot and RT-PCR results showed that the expression of proteins, such as CD68 and TNF-α, associated with M1 macrophages was suppressed.
The activation of M2 macrophages play an important role in the process of wound repair and reconstruction. They inhibit collagen synthesis and cross-linking through the synthesis of collagenase and thereby reduce collagen fibrosis and wound shrinkage [31]. The expression of iNOS is directly related to the activation of M1 macrophages. Arginase-1, MHC-II, and FIZZ-1 are important inducers of M2 macrophage activation. RT-PCR and Western blot showed that the expression of iNOS was significantly reduced, while the expression of arginase-1, MHC-II, and FIZZ-1 was significantly increased. These findings indirectly indicate that the activation of M2 macrophages was relatively increased.
In the experimental group, the regulation of these factors by AgNPs indirectly regulated inflammation. These findings have increased our confidence in the efficacy of AgNPs. However, they also indicate that AgNPs will have many unknown reactions with the immune system in the body. The molecular mechanisms of their activity need to be further investigated.