4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury in mice

Abstract

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success in mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns in mice (10-week-old C57BL/6 J male mice weighing 25 ± 3 g). We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. We demonstrated increased intracellular calcium effects of 4-AP through Orai1-pSTAT6 signaling, where 4-AP significantly mitigated inflammatory effects by promoting M2 macrophage differentiation in in-vitro macrophages and post-skin burn tissues. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in mice treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

Fig. 1. 4-AP expedited burn injury closure and enhanced skin regeneration.

A Schematic illustration of experimental design to test the beneficial therapeutic effect of 4-AP in the C57BL/6 mouse burned model. B Representative digital images of burn healing in saline and 4-AP (2 mg/kg/IP, single dose immediately after surgery and until day 21) treated mice at 0, 3, 5, 7, 10, 14, 18, and 21 days post-burning. Scale bar = 5 mm. C Percent burn wound closure at each time point relative to the initial burn wound area in control and 4-AP-treated mice. n = 6 animals per group, with one burn wound per animal. D Representative images of H&E-stained sections of full-thickness burn of saline and 4-AP treated skin tissue on days 3, 7, and 21. Scale bar = 100 µm, n = 4 skin tissues per group. E, F Quantification of burn wound length and area in H&E-stained skin burn sections using ImageJ software on days 3, 7, and 21. n = 4 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, and **P < 0.0021 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 2. 4-AP attenuated pro-inflammation and accelerated anti-inflammatory effects following skin burn.

A, B Representative images of IF staining of IL1β (pro-inflammatory) and CD206 (anti-inflammatory) and its quantitative results where 4-AP significantly controlled inflammatory resolution compared to saline-treated skin burn tissues on days 3 and 7. Scale bar = 100 µm. n = 4 skin tissues per group. C qRT-PCR data shows that 4-AP treatment significantly attenuated pro-inflammatory genes (IL1β and TNFα; M1 macrophage markers) and upregulated anti-inflammatory genes (CD206, ARG-1, and IL10; M2 macrophage markers) compared to the saline group on days 3 and 7. n = 3 skin tissues per group. D, E Western blotting images and quantitative results show that 4-AP treatment significantly attenuated IL1β and upregulated CD206 and ARG-1 protein expressions compared to the saline group on days 3 and 7. n = 3 skin tissues per group. Data are represented as mean ± SEM, and the statistical significance is indicated by asterisks (*P < 0.05 and **P < 0.0021 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 3. 4-AP augmented macrophage reparative function via Orai1 calcium channel signaling.

A, B Representative images and quantitative results of IF staining of calcium signaling in control, LPS, and LPS + 4-AP treated mouse bone marrow-derived macrophages (BMMØs) at 24 h. Scale bar = 100 µm. n = 4 per group. C qRT-PCR data shows that LPS + 4-AP treatment significantly increased the expression of Orai1 and STAT6 in BMMØs compared to the LPS and control groups. n = 3 per group. D, E Western blotting images and quantitative results showed that LPS + 4-AP treatment significantly increased Orai1 and STAT6 protein expressions in BMMØs compared to the LPS and control groups. n = 3 per group. F, G Representation of flow cytometry histogram and a quantitative result of percent CD206^+ve macrophages gated against F4/80^+ve cells in LPS versus LPS + 4-AP treated groups. n = 3 per group. H qRT-PCR data shows that 4-AP treatment significantly attenuated pro-inflammatory genes (IL1β and TNFα) and augmented anti-inflammatory genes (IL10 and CD206) in BMMØs under LPS stress conditions. n = 3 per group. I qRT-PCR data shows that 4-AP treatment significantly increased expression of Orai1 and STAT6 genes in 4-AP treated skin burn tissues on days 3 and 7. n = 3 skin tissues per group. J, K Western blotting images and quantitative results show that 4-AP treatment significantly increased Orai1 and STAT6 protein expressions compared to the saline group on days 3 and 7. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and ***P < 0.0002 vs. control/LPS/saline group) and compared using two-tailed, unpaired t-tests for two groups and ordinary one-way ANOVA for three groups.

Fig. 4. 4-AP enhanced angiogenesis following skin burn.

A, B Representative images and quantitative results of IF staining of CD31 in saline and 4-AP treated skin burn tissues on days 3 and 7. Scale bar = 100 µm. n = 4 skin tissues per group. C qRT-PCR data shows that 4-AP treatment significantly increased the expression of angiogenesis genes (VEGF, CD31, and eNOS) compared to the saline group on days 3 and 7. n = 3 skin tissues per group. D, E Western blotting images and quantitative results showed that 4-AP treatment significantly increased angiogenesis protein expressions (VEGF, CD31, and eNOS) compared to the saline group on days 3 and 7. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and ***P < 0.0002 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 5. 4-AP attenuated pro-apoptosis and accelerated anti-apoptosis effects following skin burn.

A, B Representative images of DAB-TUNEL staining of apoptosis and quantitative results of TUNEL positive cells in saline and 4-AP treated skin burn tissues on days 3 and 7. n = 4 skin tissues per group. C qRT-PCR data shows that 4-AP treatment significantly attenuated pro-apoptosis genes (BAX, caspase-9, and caspase-3) and upregulated anti-apoptosis genes (BCL2) compared to the saline group on days 3 and 7. n = 3 skin tissues per group. D, E Western blotting images and quantitative results showed that 4-AP treatment significantly attenuated expressions of pro-apoptosis protein (BAX) and upregulated anti-apoptosis proteins (BCL2 and BCL-XL) as compared to the saline group on days 3 and 7. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and ***P < 0.0002 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 6. 4-AP accelerated epithelization and keratinocytes terminal differentiation following skin burn.

A, B Representative images and quantitative results of IF staining of keratinocyte markers (K10; terminal differentiation and K14; proliferative) in saline and 4-AP treated skin burn tissues on days 3 and 7. Scale bar = 100 µm. n = 4 skin tissues per group. C, D Western blotting and (E) qRT-PCR quantitative results show that 4-AP treatment significantly increased keratin markers protein and gene (K10 and K14) expressions compared to the saline group on days 3 and 7. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and ***P < 0.0002 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 7. 4-AP increased fibroblast to myofibroblast transformation following skin burn.

A, B Representative images and quantitative results of IF staining of vimentin (fibroblasts) and α-SMA (myofibroblasts) in saline and 4-AP treated skin burn tissues on days 3 and 7. Scale bar = 100 µm. n = 4 skin tissues per group. C qRT-PCR data shows that 4-AP treatment significantly increased the expression of TGFβ, vimentin, α-SMA, and FGF genes compared to the saline group on days 7 and 21. n = 3 skin tissues per group. D, E Western blotting images and quantitative results showed that 4-AP treatment significantly increased the expression of TGFβ, vimentin, and α-SMA proteins compared to the saline group on days 7 and 21. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and ***P < 0.0002 vs. saline group) and compared using two-tailed, unpaired t-tests.

Fig. 8. 4-AP advanced skin burns wound repair by increasing collagen and MMP expressions.

A, B Representative images and quantitative results of Herovici’s staining of collagen III/I in saline and 4-AP treated skin burn tissues on days 7 and 21. n = 4 skin tissues per group. C qRT-PCR data shows that 4-AP treatment significantly increased the expression of collagen-III/I and MM9/3 genes compared to the saline group on days 7 and 21. n = 3 skin tissues per group. D, E Western blotting images and quantitative results showed that 4-AP treatment significantly increased collagen-III/I protein expressions compared to the saline group on day 21. n = 3 skin tissues per group. Data are represented as mean ± SEM. The statistical significance is indicated by asterisks (*P < 0.05, **P < 0.0021, and P*** < 0.0002 vs. saline group) and compared using two-tailed, unpaired t-tests. Schematic illustration of early (F) and late (G) phases of skin burn healing. 4-AP expedites burn wound healing via controlling multi-cellular events of (i) macrophage, (ii) apoptosis, (iii) angiogenesis, (iv) keratinocyte, and (v) fibroblast. During the early phase (days 3 and 7) of burn wound healing, 4-AP significantly mitigates inflammation via Orai1-pSTA6 signaling and apoptosis and enhances angiogenesis, keratin, and fibroblast differentiation. In the later phase (day 21), 4-AP promotes wound closure by accelerating keratinocyte differentiation and accelerating the remodeling of the ECM via stimulating fibroblast and collagen differentiation.