MLKL are involved in the regulation of skin wound healing and the interplay between macrophages and myofibroblasts

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It is widely recognized that MLKL-dependent necrosis is closely related to inflammation in wound healing, which contributes to detrimental pathologies. In present study, transcriptome sequencing data indicate sustained overexpression of MLKL throughout the wound healing process, extending beyond the early inflammation phase. In vivo experiments clearly demonstrate that MLKL deficiency delays skin wound healing, as evidenced by morphological observations and pathological characteristics. MLKL deficiency impairs the synthesis of inflammatory factors (IL-6, TNF-α, and PGE 2 ) and tissue repair-related molecules (EGF, VEGF, ERα, and MMP-9) at the wound site, potentially leading to delayed wound closure. Furthermore, we have identified the roles of MLKL and PGE 2 in the interaction between macrophages (both classically activated and alternatively activated) and myofibroblasts, an interaction essential at each stage of wound healing. Our findings suggest that MLKL's involvement in wound healing may not solely rely on necrosis-induced inflammatory responses during the early stages but also contributes to other activities in tissue regeneration. Biological sciences/Immunology Biological sciences/Molecular biology Skin Woud healing MLKL Macrophages Myofibroblasts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The skin, largest organ in the body, safeguards normal physiological activities and internal organs from environmental mechanical, microbial, or chemical stimuli. Constantly exposed to various external challenges, the skin frequently incurs wounds. An evolutionarily conserved process exists to close wounds, comprising hemostasis, inflammation, cellular proliferation, and remodeling 1 . Wound healing is typically regulated by a complex system of chemokines, cytokines, and tissue growth factors, alongside interactions between different cell types at the wound site 2 . Impairments or delays at any step in these processes can result in prolonged wound healing 2 . A comprehensive understanding of the skin wound healing process at the molecular level is essential for enhancing therapeutic strategies for wound management. Skin tissue regeneration requires the collaboration and crosstalk of various cell types and multiple mediators from the onset. After injury, macrophages are recruited to the wound site, following or simultaneously with neutrophil invasion 3 . Macrophages are highly efficient in tissue repair due to their well-known versatility and high plasticity 4 . They phagocytose necrotic cellular debris and pathogenic material or microorganisms at the injury site through evolutionarily conserved receptors. Additionally, macrophages exhibit morphological changes and other actions in response to local signals 3 . Traditionally, macrophages are categorized into two main subsets: classically activated (M1 macrophages) and alternatively activated (M2 macrophages). M1 macrophages are induced by pro-inflammatory stimuli and further propagate inflammation by releasing inflammatory cytokines (e.g., IL-1, IL-6, and TNF-α). As wound healing progresses, these macrophages phagocytose apoptotic neutrophils, replacing them as the primary inflammatory cells. During the later stages of wound healing, M2 macrophages are responsible for releasing growth factors that promote angiogenesis, re-epithelialization, and fibroplasia 5 . The ablation of macrophages leads to delayed wound repair, directly evidencing their crucial role in wound healing 6 . Similarly, delayed re-epithelialization and angiogenesis are observed when macrophages are knocked down during early healing stages 3 . Macrophages in the wound bed can activate proliferation in fibroblasts 7 . After injury, multiple subsets of fibroblasts become activated myofibroblasts, which contribute to tissue repair and scar formation 7 . Fibroblasts produce extracellular matrix (ECM) molecules that regulate tissue strength and resilience. An imbalance in ECM maintenance can lead to tissue dysfunction 8 . The cross-talk between different cell types is crucial in the four overlapping phases of wound healing, though the underlying mechanisms are not yet fully elucidated. Cytokines are essential intermediates in the cross-talk between different cell types during wound healing. There are two primary modes of cytokine release: secretion from intact cells and extravasation from necrotic cells. Necroptosis, typically characterized by eventual cell lysis accompanied by the release of numerous cytokines or damage-associated molecular patterns (DAMPs), induces inflammatory responses 9 . Necroptosis is abundant in wounds and is recognized as a significant factor affecting wound healing through the induction of inflammatory responses 10 . Genetic studies have demonstrated that necroptosis is pro-inflammatory in vivo 11 . Necroptosis relies on the activity of the pseudokinase mixed lineage kinase domain-like (MLKL) protein 12 . Upon activation, MLKL forms oligomers and translocates to the plasma membrane, inducing cell death 13 . In mice, skin inflammation can be suppressed by the deletion of MLKL 14 . Receptor-interacting protein kinase 3 (RIPK3), the upstream regulator of MLKL, also contributes to the progression of wound healing 2 . Previous literature suggests that MLKL might play a role in tissue repair by regulating inflammation or other processes. However, direct evidence that MLKL engages in the skin wound healing process has yet to emerge. In the present study, transcriptome sequencing was employed to identify potential genes related to wound healing, with MLKL identified as a target in regulating this process. We used Wild-type (WT) C57BL/6J mice and MLKL-deficient (MLKL −/− ) mice as experimental models to examine the roles of MLKL in skin wound healing by creating dorsal cutaneous wounds. The roles of MLKL in the interaction between macrophages (M1 and M2) and myofibroblasts were investigated. Furthermore, we identified a potential intermediary, PGE 2 , responsible for mediating the roles of MLKL in regulating the interaction between M1/M2 macrophages and myofibroblasts. Results Transcriptome sequencing results: MLKL expression in skin wound site To identify potential target genes associated with tissue repair, particularly those related to necrosis, we conducted transcriptome sequencing at the wound site of C57BL/6J mice from day 0 to day 14. Based on the transcriptome sequencing data, we selected genes associated with programmed cell death and mapped the top 30 upregulated genes. We identified four genes with sustained overexpression throughout the wound healing process from day 0 to day 14: Mlkl, Sycp2, Hk3, and Tnf (Fig. 1 B). Subsequently, we used RT-PCR to assess MLKL mRNA expression levels during wound healing from day 0 to day 14. The RT-PCR results confirmed that MLKL overexpression was consistently observed in the wound tissue from day 0 to day 14 (Fig. 1 C). Together, these findings suggest that MLKL may serve as a potential regulator in the skin wound healing process. Morphological results: MLKL deficiency delays skin wound healing To investigate the involvement of MLKL in skin wound healing in mice, the process of skin wound closure was compared between C57BL/6J mice and MLKL −/− mice within 14 days. The results showed that delayed wound healing could be observed in MLKL −/− mice, as compared with C57BL/6J mice, which was obvious on 3rd, 5th, 7th, and 10th days (Figure. 2A) . The data of wound area ratio was presented in Table 1 . As shown in Figure. 2B , the histological characteristics in the process of wound closure indicated that the healing speed of skin wound was impaired in MLKL −/− mice. On day 3, necrotic scab was observed on the surface of the wounds of both mice, with a large amount of serous fluid and cellulose exudation, and granulation tissue regeneration at the edge of the wounds. The difference was that the epidermis at the edge of the wound in C57BL/6J mice began to regenerate and grow into the wound, while this phenomenon did not occur at the edge of the wound in MLKL −/− mice. On day 5, the surface of wounds in both groups was covered with a thick layer of necrotic scabbed, and the granulation tissue filled the wounds and began to mature, with epidermal differentiation and thickening. The difference was that more granulation tissue was filled in the wounds of C57BL/6J mice than MLKL −/− mice, and epidermal regeneration covered half of the wounds in C57BL/6J mice and 1/3 of the wounds in MLKL −/− mice. On day 7, the epidermis on the wound surface of the two kinds of mice regenerated completely, forming a thick layer covering the wound. The wound was filled with granulation tissue regeneration, and most of the granulation tissue was mature. The difference was that the granulation tissue near the regenerated epidermis in the wound of C57BL/6J mice was obviously congested. Between granulation tissue and epidermis regeneration in the wounds of MLKL −/− mice, local granulation tissue was congested and blood vessels were dilated. On day 10, the granulation tissue filled the wounds in the two kinds of mice, most of which were mature. The epidermis was completely regenerated and well differentiated, and the granulation tissue near the regenerated epidermis was obviously congested. The difference was that the collagen fibers in the wounds of C57BL/6J mice were significantly more than those of MLKL −/− mice. On day 12, granulation tissue filled the wounds of the two kinds of mice, and the epidermis regenerated completely and differentiated well to form thick epidermis. Most of the granulation tissue in the wounds of C57BL/6J mice was mature, with increased collagen fibers and local vascular congestion. Most granulation tissue in the wounds of MLKL −/− mice began to mature, and scattered neutrophils were infiltrated in granulation tissue. On day 14, the granulation tissue in the wounds of both mice was filled and fully mature, and the epidermal regeneration completely covered the wounds to form a thick layer. These findings suggested that MLKL could be indispensable in re-epithelialization and wound contraction of skin wound healing in mice. Table 1 Circular excision wound area (mm 2 ) on various days after injury Data are presented as the mean ± SD. Differences with P values < 0.05 were considered statistically significant (* P < 0.05, *** P < 0.001). n = 6 animals in each group. Groups Wound area (mm 2 ) ± SD 0 3 5 7 10 12 14 C57BL/6J 28.26 ± 0.0 24.71 ± 0.11 16.84 ± 0.42 11.82 ± 0.74 4.60 ± 0.1 2.87 ± 0.13 0.068 ± 0.08 MLKL −/− 28.26 ± 0.0 25.86 ± 0.03* 18.51 ± 0.46*** 14.4 ± 0.18*** 6.63±*** 3.39 ± 0.31 0.344 ± 0.06 MLKL deficiency decreases inflammatory response in wound tissue. It is known that MLKL regulates necrosis, and necrosis is closely related to inflammatory response in wound site after injury 15 . The necrosis related factors and inflammatory mediators was detected in wound tissue of C57BL/6J and MLKL −/− mice. The immunofluorescent staining showed that the protein expression of Caspase-3 and P53 in MLKL −/− wound area was lower than in C57BL/6J mice (Figure. 3A) . The protein expression of B Cell Leukemia 2 (Bcl-2) in MLKL −/− wound area was higher than in C57BL/6J mice (Figure.3A) . Above results indicated that MLKL deficiency impaired the cell death in wound area. Second, the inflammatory mediator including IL-6, TNF-α and prostaglandin E 2 (PGE 2 ) secretion in MLKL −/− wound tissue was lower than in C57BL/6J mice (Figure. 3B) . For serum, IL-6 and TNF-α secretion in MLKL −/− wound tissue was lower than in C57BL/6J mice. The PGE 2 in serum of MLKL −/− was higher than that in C57BL/6J mice (Figure. 3B) . Taken together, these results suggested that MLKL was involved in regulating necrosis and inflammatory response in wound healing process. MLKL deficiency impairs tissue growth factor expression in wound site Tissue growth factors play a crucial role in wound healing. We examined the expression of growth factors, including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), estrogen receptor α (ERα), and matrix metalloprotein-9 (MMP-9), in the wound tissue of both C57BL/6J and MLKL −/− mice. Fluorescence staining revealed that the protein expression levels of EGF, VEGF, ERα, and MMP-9 were lower in the MLKL −/− wound area compared to C57BL/6J mice (Figure. 4) . These findings suggest that MLKL is involved in regulating the synthesis of growth factors at the skin wound site. MLKL in M1/M2 macrophages regulates the activity of myofibroblasts Macrophages activate myofibroblasts proliferation and its biological activity during the mid-stage of wound healing 7 . We co-cultured M1ø CM withC57BL/6J myofibroblasts. The results indicated that M1 macrophages could induce ERα, VEGF and MMP-9 expression in myofibroblasts. To explore the roles of MLKL in M1 macrophages in regulating the activity of myofibroblasts, C57BL/6J and MLKL −/− M1ø CM was co-cultured withC57BL/6J myofibroblasts separately. We found that the inducible effect of M1ø CM on growth factors expression in myofibroblasts was inhibited when treatment replaced with MLKL −/− M1ø CM (Figure. 5A) . These results suggested that MLKL in M1 macrophages could regulate the growth factor expression in myofibroblasts. Similarly, we co-cultured M2ø CM withC57BL/6J myofibroblasts. The results demonstrated that M2ø CM could induce ERα, VEGF and MMP-9 expression in myofibroblasts. To explore the roles of MLKL in M2 macrophages in regulating the activity of myofibroblasts, C57BL/6J and MLKL −/− M2ø CM was co-cultured with C57BL/6J myofibroblasts separately. We found that the inducible effect of C57BL/6J M2ø CM on growth factors expression in myofibroblasts was inhibited when treatment replaced with MLKL −/− M2ø CM (Figure. 5B) . These results suggested that MLKL in M2 macrophages could regulate the growth factor expression in myofibroblasts. MLKL in fibroblasts regulate the activity of M1/M2 macrophages To investigate the effects of myofibroblasts on the activity of macrophages. We co-cultured C57BL/6J MFbCM with M1 and M2 macrophages separately. The results indicated that C57BL/6J MFbCM induced the synthesis of IL-6, nitric oxide (NO), and TNF-α in M1 macrophages, whereas this inductive effect was inhibited when MLKL −/− MFbCM was used instead (Figure. 6A) . This suggests that MLKL in myofibroblasts regulates the activity of M1 macrophages. Ym1 and arginase are primarily expressed in macrophages at the site of injury 16 . We observed lower expression levels of arginase and Ym1 in the MLKL −/− skin wound site compared to the C57BL/6J wound site (Figure. 6C) . Additionally, the results showed that C57BL/6J MFbCM induced the synthesis of IL-10, arginase, and Ym1 in M2 macrophages, whereas this inductive effect was inhibited when MLKL −/− MFbCM was used (Figure. 6B) . This suggests that MLKL in myofibroblasts also regulates the activity of M2 macrophages. PGE 2 is one of the mediators in the cross-talk between macrophages and myofibroblasts The results above indicate that MLKL plays a role in the interaction between macrophages and myofibroblasts. We observed that the expression of cyclooxygenase-2 (COX-2) and the secretion of PGE 2 were lower in MLKL −/− M1/ M2 macrophages and myofibroblasts compared to C57BL/6J cells (Figure. 7) , suggesting that PGE 2 might serve as an intermediary in the interaction between macrophages and myofibroblasts. To explore this further, we supplemented MLKL −/− MFbCM or M1ø / M2ø CM with exogenous PGE 2 to match the amount lost in MLKL −/− cells. The results demonstrated that MLKL −/− M1ø CM or M2ø CM supplemented with PGE 2 enhanced the expression of VEGF and MMP-9 in myofibroblasts ( Fig. 8 A and 8 B ) . However, when MLKL −/− MFbCM was supplemented with PGE 2 , the effects differed: IL-6 synthesis remained unaffected, NO secretion was further inhibited, and TNF-α was enhanced in M1 macrophages ( Fig. 9 A ) . Additionally, MLKL −/− MFbCM supplemented with PGE 2 decreased IL-10 and Ym1 expression, while enhancing arginase expression in M2 macrophages ( Fig. 9 B ) . Therefore, these findings suggest that the roles of PGE 2 in inducing M1/M2 macrophage activation by myofibroblasts are more complex than those in inducing myofibroblast activation by M1/M2 macrophages. Discussion MLKL-dependent necrosis is commonly associated with adverse pathologies; however, our study reveals that MLKL is essential for normal skin wound healing. In vivo experiments demonstrate that MLKL deficiency results in delayed wound repair. While MLKL is traditionally thought to affect wound healing through modulation of necrosis-induced inflammation, our findings suggest that its role extends beyond this mechanism. Firstly, MLKL overexpression persists throughout the entire wound healing process, from incision to closure, rather than being restricted to the inflammation phase. Secondly, MLKL deficiency negatively impacts both the wound healing rate and the expression of growth-related factors. Thirdly, the regulatory effect of macrophages on myofibroblast activity is diminished in the absence of MLKL. Conversely, while myofibroblasts can activate macrophages, this activation is partially inhibited by MLKL −/− myofibroblasts. Our study also establishes that PGE 2 functions as an intermediary in the interaction between macrophages and myofibroblasts. In summary, MLKL plays a crucial role in skin wound healing by mediating a complex network of cellular interactions and cytokine expressions, thereby contributing to the establishment of a tissue repair microenvironment at the wound site. The initial damage stimulus induces a conformational change in cells at the wound site, leading to plasma membrane translocation and the lethal permeation of the lipid bilayer. This process results in the release of cellular contents and the subsequent activation of an inflammatory response 17 . Pro-inflammatory chemokines and cytokines released by activated macrophages, primarily M1 macrophages, initiate the recruitment and activation of additional immune cells, including neutrophils and bone marrow-derived monocytes, to the injury site. These immune cells further amplify inflammation to eliminate damage signals and necrotic cells 18 . Necroptosis is mediated by RIPK3 19 . RIPK3 deficiency significantly delays wound closure and impairs wound healing quality, as evidenced by delayed re-epithelialization, angiogenesis, granulation tissue formation, and collagen deposition 14 . MLKL, a functional substrate for RIPK3, acts as an adaptor protein in necrosis signal transduction 20 . Both RIPK3 and MLKL overexpression are observed at early stages of skin wound healing. Additionally, three other targets (Sycp2, HK3 and TNF-α) are overexpressed at the wound site. Sycp2, a synaptonemal complex protein, is associated with meiosis 21 . HK3 is essential for initiating glycolysis 22 . Both Sycp2 and HK3 may be involved in cellular differentiation related to inflammatory and immune responses 22 . TNF-α is crucial in both apoptosis and necroptosis, and its interaction with MLKL in necroptosis is well-documented 23 . Therefore, the co-overexpression of TNF-α and MLKL could be observed at wound sites as expected. In MLKL −/− mice, delayed skin wound closure and impaired morphological characteristics throughout the healing process are observed. Additionally, the concentration of inflammatory cytokines, including PGE 2 , TNF-α, and IL-6, is lower in the wound tissue of MLKL −/− mice compared to C57BL/6J mice three days post-injury. Serum levels of TNF-α and IL-6 are also reduced in MLKL −/− mice, while PGE 2 levels are elevated in their serum after injury. The wound site data are likely more indicative of the local inflammatory microenvironment than serum data, which reflects a systemic rather than a localized response 24 . MLKL may reduce the inflammatory response at the wound site through two potential mechanisms: it could directly regulate the synthesis of inflammatory cytokines, leading to decreased intracellular cytokine production in MLKL −/− mice. Alternatively, reduced cell death, as evidenced by altered expression of P53, Bcl-2, and Caspase-3, could result in less cellular content release, potentially reducing inflammatory cytokine release in MLKL −/− wound sites. It is possible that both mechanisms contribute to the reduced inflammatory cytokine release observed in MLKL −/− skin wounds. Overall, these results suggest that MLKL deficiency impairs skin wound healing, likely due to diminished inflammatory responses at the wound site, particularly during the early stages of healing. As wound healing progresses from the pro-inflammatory to the pro-healing phase, it transitions into the remodeling stage. This stage is marked by a shift in macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) 25 . The debate continues on whether M2 macrophages at injury sites arise from blood monocytes or result from the phenotypic conversion of M1 macrophages 4 . These anti-inflammatory macrophages are crucial for inflammation resolution and the progression of remodeling. They contribute to the healing process through their regenerative properties by secreting angiogenic and growth factors, cytokines, and chemokines, such as metalloproteinases (MMPs), VEGF, IL-8, TGF-β, IL-10, and arginase, which are essential for recruiting and activating other cells. Depletion of macrophages during the regenerative phase can directly or indirectly impact wound revascularization, matrix production, and re-epithelialization 16 . Our study reveals that MLKL and RIPK3 are sustained in the wound area during mid- and late-stages of healing, indicating that RIPK3-MLKL signaling may be important not only in the early inflammatory phase but also in the later stages of wound repair. We found that MLKL deficiency impairs the expression of growth-related factors, such as EGF, VEGF, MMP-9, and ERα, during the mid- and late-stages of wound healing. This suggests that MLKL plays a role in regulating growth factor expression at the wound site during the later stages of healing. The delayed wound regeneration observed in MLKL −/− mice, as evidenced by H&E staining, aligns with these findings. Specifically, collagen fibers and mature granulation tissue were significantly more abundant in wounds of C57BL/6J mice compared to MLKL −/− mice at later stages of healing. Miscommunication between macrophages and fibroblasts is recognized as a critical factor that can shift the balance from physiological repair to pathological fibrosis 8 . It is well-documented that macrophage depletion during wound healing leads to a reduction in fibroblast numbers and that macrophages play a role in scavenging TGF-β, a key regulator of fibroblast/myofibroblast activation 18 . Supernatants from C57BL/6J M1 macrophages were found to enhance the expression of VEGF and MMP-9 in fibroblasts. This effect was partially inhibited when supernatants from MLKL −/− M1 macrophages were used instead, indicating that MLKL in macrophages may regulate myofibroblast activity. Additionally, PGE 2 synthesis, which is known to increase at both early and late stages of wound healing 26 . We hypothesized that PGE 2 is a potential mediator in the cross-talk between M1 macrophages and myofibroblasts, as we found that COX-2 (the upstream enzyme of PGE 2 synthesis) expression and PGE 2 levels in MLKL −/− wound tissue were lower than in C57BL/6J wound tissue (Figure. 7) . To investigate this further, we replenished PGE 2 in a co-culture system of MLKL −/− M1 macrophages and myofibroblasts. This restoration of PGE 2 led to a recovery in the expression patterns of VEGF and MMP-9 in myofibroblasts. Conversely, MLKL in myofibroblasts was found to regulate the synthesis of IL-6, NO, and TNF-α in M1 macrophages through PGE 2 . These findings suggest that PGE 2 may act as an intermediary in the MLKL-mediated interaction between M1 macrophages and myofibroblasts. Regarding the mechanism behind the reduced PGE 2 concentration in supernatants from MLKL −/− macrophages (both M1 and M2) and myofibroblasts, two potential explanations exist: MLKL deficiency might directly reduce PGE 2 synthesis in these cells, or it might delay membranolysis, trapping PGE 2 within the cells. Our data, particularly the reduced COX-2 expression in MLKL −/− cells, support the first possibility. Taken together, MLKL may play a crucial role in maintaining the early-stage inflammatory microenvironment at the wound site by modulating the interaction between M1 macrophages and fibroblasts. M2 macrophages produce anti-inflammatory cytokines and growth factors that mitigate inflammation, activate fibroblasts, and promote angiogenesis and wound contraction 18 . It has been demonstrated that ablation of macrophages reduces myofibroblast numbers in the wound bed, impairs myofibroblast function, and hinders wound healing 6 , 7 . Prolonged pathological stimulation leading to unresolved crosstalk between M2 macrophages and fibroblasts often results in pathological fibrosis or chronic wounds 27 . Thus, understanding the interaction between M2 macrophages and fibroblasts is essential. We observed that the expression of growth factors EGF, VEGF, and MMP-9 peaks at the late stage of wound healing; however, their levels were lower in MLKL −/− wound sites compared to C57BL/6J sites. MLKL in M2 macrophages appears to regulate VEGF and MMP-9 expression in myofibroblasts, suggesting that MLKL deficiency might lead to reduced growth factor expression at the wound site. Additionally, MLKL in fibroblasts can regulate the expression of arginase, Ym1, and IL-10 in M2 macrophages, indicating that MLKL in fibroblasts may also influence M2 macrophage activity (Figure. 6) . Ym1 and arginase, which are predominantly expressed in macrophages at injury sites, play crucial roles in wound healing and resolution 16 . Arginase contributes to wound healing through the production of L-ornithine, with its absence impairing cutaneous wound healing and its overproduction contributing to fibrosis 28 . IL-10 promotes the differentiation of macrophages towards pro-regenerative phenotypes 29 . PGE 2 released from myofibroblasts, along with its receptor agonist, enhances arginase activity in M2 macrophages, further supporting the role of PGE 2 in regulating M2 macrophage activity 30 . We also found reduced COX-2 and PGE 2 synthesis in MLKL −/− M2 macrophages. Similar to its role in the interaction between M1 macrophages and myofibroblasts, PGE 2 is involved in the interaction between M2 macrophages and myofibroblasts. These findings highlight the importance of MLKL and PGE 2 in mediating the complex interactions between macrophages and fibroblasts during wound healing (Figure. 10) . In conclusion, this study provides direct evidence that MLKL plays a crucial role in skin wound healing by modulating the wound site's microenvironment. We have also elucidated the involvement of MLKL in the interaction between macrophages and fibroblasts, identifying PGE 2 as a potential intermediary in the MLKL-mediated communication between both M1 and M2 macrophages and fibroblasts. Material & Method Animals The mice experiment of present study were performed under the guidance of the Regulations for the Administration of Affairs Concerning Experimental Animals in China (2017). The experimental design was approved by the Animal Welfare and Research Ethics Committee of the university (approval ID: NND2022021). C57BL/6J mice were provided by the Model Animal Research Center of Nanjing University, Nanjing, China. MLKL −/− mice were gifted by Professor Shuixing Yu of Inner Mongolia Agricultural University, who has previously done related research 31 . The mice were housed in a clean environment (12 h day/night cycles, 22–24 ℃ and 50% humidity) supplied with unlimited food and water. Mice at age eight-weeks-old (25-30g) were used in all experiments. This study was conducted in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ). Experimental design The in vivo study, transcriptome sequencing and analysis of wound sites from C57BL/6J mice were conducted to identify potential genes involved in skin wound healing (Figure S1 A). To investigate the roles of MLKL in this process, we created skin wounds in both C57BL/6J and MLKL −/− mice. The wound healing ratio, morphological characteristics, and expression of tissue repair-related factors at the wound site were monitored throughout the healing process using H&E staining and immunofluorescence staining (Figure S1 A). The in vitro study, M1/M2 macrophages and myofibroblasts were used to investigate the roles of MLKL in the interaction between these cell types. We co-cultured M1 macrophage conditioned medium (M1ø CM) or M2ø CM withC57BL/6J myofibroblasts as previously described 30 . The expression of ERα, VEGF, and MMP-9 in myofibroblasts was detected. Further, to explore the roles of MLKL in M1/M2 macrophages in regulating myofibroblast activity, C57BL/6J and MLKL −/− M1ø CM (or MLKL −/− M2ø CM) were co-cultured withC57BL/6J myofibroblasts separately. The expression of ERα, VEGF, and MMP-9 in myofibroblasts was assessed by Western blotting and RT-PCR. To investigate the effects of myofibroblasts on M1/M2 macrophages, we co-cultured myofibroblast conditioned medium (MFbCM) with M1/M2 macrophages separately. The expression of IL-6, nitric oxide (NO), and TNF-α in M1 macrophages was measured, as well as IL-10, arginase, and Ym1 in M2 macrophages. Additionally, to explore the roles of MLKL in myofibroblasts in regulating the activity of M1/M2 macrophages, C57BL/6J and MLKL −/− MFbCM were co-cultured with M1/M2 macrophages separately. The expression of IL-6, NO, TNF-α and IL-10 was detected by ELISA, arginase and Ym1 expression in M2 macrophages was detected by Western blotting. Finally, the roles of PGE 2 in the MLKL-mediated interaction between macrophages and myofibroblasts were investigated. The experimental design abstract is attached (Figure S1 C, D). Cutaneous wound model The circular excisional wound was directly created on the dorsal skin of C57BL/6J and MLKL −/− mice. Briefly, the experimental mice were anesthetized with isoflurane inhalation. The dorsum was shaved and sterilized with 75% alcohol. A 6-mm diameter skin biopsy punch was used to create an excision wound extending to the fascia. Wound closure progression was measured daily until day 14 post-wound. At days 0, 3, 5, 7, 10, 12, and 14 after wound injury, mice were sacrificed following anesthesia, and skin samples were collected from the entire wound sites (including the scab and epithelial margins). The skin samples were fixed and stored in 10% formalin for histological analysis or frozen in liquid nitrogen and stored at -80°C for molecular experiments. Transcriptome analysis Total mRNA was extracted using TRIzol reagent (Invitrogen, CA, USA). Libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Differentially expressed genes (DEGs) were identified using DESeq2 and screened under the criteria of |log2 Fold Change| > 1.5 and P -value < 0.05. The identified DEGs underwent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using OECloud tools ( https://cloud.oebiotech.com/task/ ). Significant enrichment was defined by a P -value < 0.05, and the top 30 enrichment results are presented. Wound healing rate determination The wound healing rate at 0, 3, 5, 7, 10, 12, and 14 days was calculated using the wound surface area compared to the original wound area. The percentage of wound closure was determined using the formula: (AreaDay 0-AreaDay)/AreaDay 0×100%. Digital images of the wound surface were taken using a SONY Alpha 6400 camera. The wound area was quantified using ImageJ software (National Institutes of Health, MD, USA). Hematoxylin and eosin (H&E) staining The skin tissue samples fixed in 10% formalin were sectioned into 2 µm in thickness. The sections were dehydrated using a graded series of ethanol (100%, 75%, 50%, and 25%), embedded in paraffin, and stained with hematoxylin and eosin (H&E). Images of the stained sections were acquired using an Axio Scan.Z1 slide scanner (Zeiss, Oberkochen, Germany). Immunofluorescence staining The excised dorsal tissue samples from C57BL/6J mice and MLKL −/− mice were frozen in liquid nitrogen and stored at -80°C. Subsequently, the tissues were thawed and embedded in Tissue-Tek OCT compound (Sakura Finetek, CA, USA), and serial cryosections of 6 µm thickness were prepared using a freezing microtome. The sections were fixed in cold acetone for 10 minutes, washed with cold endotoxin-free phosphate-buffered saline (PBS) containing 0.25% Tween-20, and blocked for 1 hour with 3% bovine serum albumin. Primary antibodies were then added, and the sections were incubated in the dark for 14 hours at 4°C. After incubation, the slides were washed three times in PBS with 0.25% Tween-20 and incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature. DAPI (4',6-diamidino-2-phenylindole) was used for nuclear counterstaining. Fluorescence signals were captured using a confocal microscope (LSM 800, Zeiss, Oberkochen, Germany). Fields were selected randomly for imaging. The details of the antibodies used are listed in Table 2 . Table 2 Antibodies used in immunofluorescence staining p53: Tumor protein 53; Bcl-2: B-cell lymphoma 2; Caspase-3: Cysteinyl aspartate specific proteinase 3; ERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; EGF: Epidermal growth factor; VEGF: Vascular endothelial growth factor; Ym1: Chitinase-3-like Protein 1; Dnk: Donkey; pAb: polyclonal antibody; Rb: Rabbit. Name Description Concentration Company Cat No. p53 Mouse monoclonal 1:2000 Cell signaling Technology 2524T Bcl-2 Mouse monoclonal 1:50 Cell signaling Technology 15071 Caspase-3 Rabbit polyclonal 1:1000 abcam ab49822 ERα Mouse monoclonal 1:500 GeneTex GTX13538 MMP-9 Rabbit polyclonal 20ug/ml Novus NBP2-41233 EGF Rabbit monoclonal 1:500 abcam ab184266 VEGF Mouse monoclonal 200ug/ml Santa SC-7269 arginase Mouse monoclonal 1:1000 Santa SC-47715 Ym1 Rabbit polyclonal 1:50 Stemcell technologies 60130 Dnk pAb to Rat IgG Alexa Fluor® 488 1:1000 abcam ab150153 Goat pAb to Rb IgG Alexa Fluor® 647 1:1000 abcam Ab150079 Isolation and culture of bone marrow-derived macrophages (BMDMs) The BMDMs were isolated from murine femurs and tibias, cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, Excell Bio, Shanghai, China), 1.2% Glutamine (Thermo scientific, Rockford, IL, USA), 2.4% penicillin-streptomycin (Gibco, USA), combined with 20 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech, NJ, USA) at a density of 5×10 6 cells in a 6-well plate. The culture medium was replaced at 24 h interval up to 5 days. The BMDMs were treated with 1ug /mL LPS (PeproTech, NJ, USA) for 24 h to differentiate into M1 macrophages, which was identified by flow cytometry (Figure. S2 in Supplementary Material). The BMDMs were treated with 20 ng/mL IL-4 in combination with 20 ng/mL IL-13 (PeproTech, NJ, USA) for 48 h to differentiate into M2 macrophages, which was identified by flow cytometry (Figure. S2 in Supplementary Material). The M1 and M2 macrophages conditioned medium (M1ø CM and M2ø CM) were collected for further experiments. Mice skin myofibroblasts cultivation in vitro At first, the fibroblasts were isolated from C57BL/6J and MLKL −/− mice. In brief, the ear explants were minced and incubated in 0.25% trypsin-EDTA (Hyclone, UT, USA) for 1 h at 37°C to remove the epidermis. The obtained explants were placed in tissue culture plates, and migration of fibroblasts from tissue explant to dish could be observed within 2–5 days. After 2 weeks, tissue explants were removed, and fibroblasts were re-seeded at a density of 1×10 5 cells in a 6-well plate and cultured in DMEM/F12 medium supplemented with 10% FBS and 2% penicillin-streptomycin combined with 100 pg/mL transforming growth factor-β (TGF-β, PeproTech, NJ, USA). The medium was replaced every 24 h until the fifth day when the fibroblasts were induced to myofibroblasts. Subsequently, the culture medium was replaced with DMEM/F12 medium supplemented with 10% FBS and 2% penicillin-streptomycin, and cultured for 2 days to remove TGF-β. The myofibroblasts were identified by α-smooth muscle actin (SMA) expression using immunofluorescence assay (Figure. S3 in Supplementary Material). The myofibroblasts conditioned medium (MFbCM) was collected for further experiments. Western blot analysis (WB) For total cellular protein extraction, cells were treated with the Mammalian Protein Extraction Reagent (Thermo scientific, Rockford, IL, USA). The protein concentrations in the samples were quantified using the BCA Assay Kit (Thermo scientific, Rockford, IL, USA). For Western blot analysis, 10 µg of total protein per lane were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. Protein bands were visualized using the Chemiluminescent Substrate (Thermo scientific, Rockford, IL, USA). Grayscale values were quantified using ImageJ software (National Institutes of Health, MD, USA). Primary antibodies, as detailed in Table 3 . Table 3 Antibodies used in western blot ERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; Ym1: Chitinase-3-like Protein 1; COX-2, Cyclooxygenase-2. Name Description KDa Concentration Company Cat No. ERα Mouse monoclonal 71.4 KDa 1:250 GeneTex GTX13538 MMP-9 Rabbit polyclonal 73 KDa 1 ug/mL Novus NBP2-41233 VEGF Rabbit polyclonal 42 KDa 1 ug/mL Novus NB100-2381 arginase Mouse monoclonal 35 KDa 1:200 Santa SC-47715 Ym1 Rabbit polyclonal 45 KDa 1:1000 Stemcell technologies 60130 GAPDH Rabbit polyclonal 37 KDa 1:5000 Affinity AF7021 COX-2 Rabbit monoclonal 70 KDa 1:1000 Cell signaling Technology D5H5 Goat Anti-Rabbit IgG (H + L) HRP / 1:5000 Affinity S0001 Goat Anti-Mouse IgG (H + L) HRP / 1:5000 Affinity S0002 Enzyme-linked immunosorbent assay (ELISA) analysis The harvested wound tissue (0.2–0.4 g) was cut into pieces, homogenized and lysed with T-PERTM Tissue Protein Extraction Reagent (Thermo scientific, Rockford, IL, USA). The blood was gained from the eyeballs and centrifuged at 3000 × g for 10 min to extract serum. The concentrations of IL-6 (Biolegend, CA, USA), TNF-α (Biolegend, CA, USA) and PGE 2 (Cayman Chemical, Ann Arbor, MI) were measured in the tissue extracts and serum. The concentration of IL-6, TNF-α, IL-10 (Invitrogen, Thermo Fisher Scientific, CA, USA) and NO (Beyotime, Shanghai, China) supernatants of cultured macrophages was measured according to the manufacturer’s instructions. Real-time RT-PCR analysis The total mRNA was extracted using the Total RNA Miniprep Kit (Axygen, California, USA) from cultured cells. The extracted RNA was reverse-transcribed into cDNA by a cDNA Reverse Transcription Kit (Vazyme, Nanjing, China). Real-time PCR reactions were conducted using the ABI QuantStudio 7 (Thermo Scientific, IL, USA). The PCR protocol was as follows: initial denaturation for 30 seconds at 95°C, followed by 35 cycles of 5 seconds at 95°C (denaturation), 34 seconds at 60°C (annealing), and 20 seconds at 72°C (elongation). GAPDH served as an internal control. Results are presented as 2 −ΔΔCt (where ΔΔCt = ΔCt – ΔCt control and ΔCt = Ct target -Ct GAPDH). The primers used in RT-PCR are detailed in Table 4 . Table 4 Primers used in this study Accession No. Gene name Primer sequence NM_008084.4 GAPDH Forward:5' -AGGTCGGTGTGAACGGATTTG-3' Reverse:3' -GGGGTCGTTGATGGCAACA-5’' NM_007956.5 ERα Forward:5'- TTCTCCCTTTGCTACGTCAC-3' Reverse:3'- ATCGCTTTGTCAACGACTTC-5' NM_013599.5 MMP-9 Forward:5'- CGCCTTGGTGTAGCACAACA-3' Reverse:3'- ACAGGGTTTGCCTTCTCCGTT-5' NM_009505.4 VEGF Forward:5'- CTGTAACGATGAAGCCCTGGAG-3' Reverse:3'- TGGTGAGGTTTGATCCGCAT-5' NM_029005.3 MLKL Forward:5'- TATGTCTCCCCTGAGAGACTGAAAA-3' Reverse:3'- TTCCCAGAGTACAATTCCAAAGCTA-5' ERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; MLKL: mixed lineage kinase domain-like protein. Data analysis All data were analyzed using GraphPad Prism 8 (GraphPad Software InC, USA) and expressed as mean ± standard deviation (SD). Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test or two-way ANOVA with Bonferroni’s post-hoc test. P < 0.05 were considered statistically significant. Declarations Acknowledgements We would like to thank Dr. Jinling Wang and Dr. Yulin Ding for their kind assistance in H&E staining. Graphic abstract of present study created with BioRender.com. Author Contributions Shuangyi Zhang, Jiamin Zhao, Bo Liu and Surong Hasi contributed to the study conception and design. Material preparation, data collection and analysis were performed by Jiamin Zhao, Yunjie Bai, Zhiguo Gong, Zhuoya Yu, Wenhui Bao and Wei Mao. The first draft of the manuscript was written by Jiamin Zhao and all authors commented on previous versions of the manuscript. Data availability The data for this study are available by contacting the corresponding authors upon reasonable request. The sequencing data of this study are openly available in GenBank of NCBI (accession No. GSE273056). Funding sources This work was supported by the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2024MS03064), National Natural Science Foundation of China (32202879), Research Program of Science and Technology at Universities of Inner Mongolia Autonomous Region (NJZZ23035). Ethics declarations Conflict of interest The authors declare that there are no competing financial interests or personal relations that might have influenced the publication of this work. Ethics approval All animal experiments were performed according to the regulations of the Administration of Affairs Concerning Experimental Animals in China. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of the Inner Mongolia Agricultural University (approval ID: NND2022021). Consent for publication All the authors have read and approved the final manuscript. 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Supplementary Files supplementalmaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 25 Oct, 2024 Reviews received at journal 24 Oct, 2024 Reviewers agreed at journal 11 Oct, 2024 Reviews received at journal 16 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor assigned by journal 08 Aug, 2024 Editor invited by journal 28 Jul, 2024 Submission checks completed at journal 25 Jul, 2024 First submitted to journal 13 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4576292","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":342329698,"identity":"1091e78a-f642-4fa8-9437-bac79a71b6ee","order_by":0,"name":"Jiamin Zhao","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiamin","middleName":"","lastName":"Zhao","suffix":""},{"id":342329699,"identity":"a3f07832-2302-4695-a214-0bf51711a185","order_by":1,"name":"Shuangyi Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCQjFAyY/GNjYkaaFcUZBWjLRWsCAmefDIcYGQjrkZ/cYfi74dVhGfkbyM2kbgwPMDOyHj27Ap8Xgzhlj6Zl9h3kMbqSZSecY3OFj4ElLu4FXi0SOgTRvD1CLRAJIyzNmBgkeM7xa5GfkGP8GaZGfkf5N2sLgMGMDIS0MN3LMpHl+HOYBMxiI0QL0Qpk1b0M6j8GZN8WWPQZpyWyE/AIMqM23ef5Y28u3p2+88eOPjR0/++Fj+B0GAoxtzQwMAgkQDhtB5WDwp46Bgf8AcWpHwSgYBaNg5AEAyIJIOCHypgYAAAAASUVORK5CYII=","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shuangyi","middleName":"","lastName":"Zhang","suffix":""},{"id":342329700,"identity":"a1721295-cb7f-42de-bea3-5064c9cb2cd7","order_by":2,"name":"Yunjie Bai","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunjie","middleName":"","lastName":"Bai","suffix":""},{"id":342329701,"identity":"ffc1926e-a3a3-437e-ab8c-0ccd802c7244","order_by":3,"name":"Zhiguo Gong","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Gong","suffix":""},{"id":342329702,"identity":"895baf4a-67d8-47e3-8d64-abfb54559e12","order_by":4,"name":"Wenhui Bao","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Bao","suffix":""},{"id":342329703,"identity":"698f64d8-8618-4d4b-8536-f7389cdeca25","order_by":5,"name":"Zhuoya Yu","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhuoya","middleName":"","lastName":"Yu","suffix":""},{"id":342329704,"identity":"70db16f2-df22-42e5-b7a0-348e7a71e997","order_by":6,"name":"Bo Liu","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Liu","suffix":""},{"id":342329705,"identity":"e1a1d74a-52ea-493b-b3ce-b498517d27f4","order_by":7,"name":"Wei Mao","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Mao","suffix":""},{"id":342329706,"identity":"e245b293-c48f-45a7-ba11-b27b4820a069","order_by":8,"name":"Surong Hasi","email":"","orcid":"","institution":"Ministry of Agriculture, Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Surong","middleName":"","lastName":"Hasi","suffix":""}],"badges":[],"createdAt":"2024-06-13 12:45:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4576292/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4576292/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-97729-2","type":"published","date":"2025-04-19T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62959431,"identity":"14d8a28c-aba6-48a6-9578-029efe7be204","added_by":"auto","created_at":"2024-08-21 13:04:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":645587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHub genes related to programmed cell death in injured tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA\u003cstrong\u003e) \u003c/strong\u003eSeven selection indices were proposed from the PCA.\u003cstrong\u003e \u003c/strong\u003eB\u003cstrong\u003e) \u003c/strong\u003eHeatmap shows top up-regulation 30 genes from programmed cell death in wound tissues from day 0 to 14. Horizontal represents genes, where each column represents one sample. Red represents increased expression genes, and blue represents decreased expression genes (n=4). C) MLKL mRNA expression in C57BL/6J skin wound tissues from day 0 to 14 was measured by RT-PCR (n=7).\u003c/p\u003e","description":"","filename":"figure.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/0a5e460915590e64e6d5d183.jpg"},{"id":62959432,"identity":"6cfefec7-ac93-47ca-a359-bc9d17e59777","added_by":"auto","created_at":"2024-08-21 13:04:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1101775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDelay of skin wound healing in MLKL\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The 6 mm diameter excisional biopsies were obtained from the back of wild type C57BL/6J mice and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice. The mean value of wound healing rate was calculated based on the original wound size of each biopsy site. The values were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test and represented mean ± SD (n = 6). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e \u003c/strong\u003eB) The representative histological examination of skin wound by H\u0026amp;E staining at day 0, 3, 5, 7, 10,12 and 14 after injury. The wound surface was marked by dashed line. Scale bar = 500 μm or 200 μm.\u003c/p\u003e","description":"","filename":"figure.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/7c572f64b93f1d6d7e1f957f.jpg"},{"id":62960179,"identity":"1524aab9-bdd4-49de-a888-30f3497f72ba","added_by":"auto","created_at":"2024-08-21 13:12:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6090710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL is involved in inflammatory mediator synthesis in wound site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The skin wound tissues of C57BL/6J and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice were subjected to immunofluorescence staining for Caspase-3 (red), Bcl-2 (green) P53 (green,) and nuclear (DAPI, blue) on the 5th, 7th and 10th days after wound injury. Merge represents the composite picture of target protein and nuclear. The confocal microscope was used for image acquisition (×200 magnification), and the mean value of fluorescence intensity was used to perform calculation (a. u, arbitrary unit). B) The secretion ofIL-6, TNF-α and PGE\u003csub\u003e2\u003c/sub\u003e in wound tissue (Day 3) and serum (Day 3) of C57BL/6J and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice was evaluated by ELISA. Results were expressed as the mean ± SD of multiple independent experiments and analyzed by one-way ANOVA followed by Tukey’s multiple-comparisons test or two-way ANOVA with Bonferroni’s post-hoc test, as appropriate (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant. a. u, arbitrary unit. Scale label = 20 μm.\u003c/p\u003e","description":"","filename":"Figure.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/4bcbfeb1f65c238f3e95b6f4.jpg"},{"id":62959436,"identity":"c03f7f41-dd90-4ee7-ab5c-dc017fdedad6","added_by":"auto","created_at":"2024-08-21 13:04:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8292992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL is involved in tissue growth related factors expression in wound site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe skin wound tissues of C57BL/6J and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice were subjected to immunofluorescence staining for MMP-9 (red), ERα (green), EGF (red) VEGF (green) and nuclear (DAPI, blue) on the 5th, 7th and 10th days after wound injury. Merge represents the composite picture of target protein and nuclear. The immunofluorescence staining was imaged by fluorescence microscopy (Zeiss LSM 800 laser, ×200 magnification), and the mean value of fluorescence intensity was used to perform calculation (a. u, arbitrary unit). Results were expressed as the mean ± SD of multiple independent experiments and analyzed by two-way ANOVA with Bonferroni’s post-test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant. Scale label = 20 μm.\u003c/p\u003e","description":"","filename":"Figure.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/d9f15a17c57080c4a45be69a.jpg"},{"id":62960175,"identity":"b56d5161-8b59-4556-bb0c-4a8bb445d73e","added_by":"auto","created_at":"2024-08-21 13:12:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":593661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL in macrophages affects the activity of myofibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The expression of VEGF, MMP-9 and ERα mRNA and protein in C57BL/6J myofibroblasts treated with C57BL/6J M1ø CM, or MLKL\u003csup\u003e-/-\u003c/sup\u003eM1ø CM. B) The expression of VEGF, MMP-9 and ERα mRNA and protein in C57BL/6J myofibroblasts treated with C57BL/6J M2ø CM, or MLKL\u003csup\u003e-/-\u003c/sup\u003eM2ø CM. GAPDH was used as a loading control (n = 3). Grayscale values were measured using ImageJ software. Results were expressed as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"Figure.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/d4fdac699d9096ae68d7d8a7.jpg"},{"id":62960762,"identity":"1f4aea3f-3231-4bc7-9219-1bf335dfbbb3","added_by":"auto","created_at":"2024-08-21 13:20:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1095979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL in myofibroblasts affects the activity of macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The secretion of IL-6, NO and TNF-α in C57BL/6J M1ø treated with C57BL/6J MFbCM, or MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM B) The secretion of IL-10, arginase and chitinase-like protein 1 (Ym1) in C57BL/6J M2ø treated with C57BL/6J MFbCM, or MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM.\u003cstrong\u003e \u003c/strong\u003eResults were expressed as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant. C)The skin wound tissues of C57BL/6J and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice were subjected to immunofluorescence staining for arginase (red), Ym1 (green) and nuclear (DAPI, blue) on the 5th, 7th and 10th days after wound injury. Merge represents the composite picture of target protein and nuclear. The immunofluorescence staining was imaged by fluorescence microscopy (Zeiss LSM 800 laser, ×200 magnification), and the mean value of fluorescence intensity was used to perform calculation (a. u, arbitrary unit). Results were expressed as the mean ± SD of multiple independent experiments and analyzed by two-way ANOVA with Bonferroni’s post-test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant. Scale label = 20 μm.\u003c/p\u003e","description":"","filename":"figure.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/6b1b6c99e1a2f53e6e4b426c.jpg"},{"id":62959433,"identity":"2b4398e2-8d22-4b27-8184-80df37eed0a1","added_by":"auto","created_at":"2024-08-21 13:04:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":414073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL affects PGE\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e synthesis in macrophages and myofibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCOX-2 and PGE\u003csub\u003e2\u003c/sub\u003e levels in M1/M2 macrophages and myofibroblasts of C57BL/6J and MLKL\u003csup\u003e-/-\u003c/sup\u003e mice. GAPDH was used as a loading control (n = 3). Grayscale values were measured using ImageJ software. Results were expressed as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"Figure.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/9f704d23c2c53e656d0b120a.jpg"},{"id":62960177,"identity":"de75d494-4695-4437-8035-1507c59e1e52","added_by":"auto","created_at":"2024-08-21 13:12:21","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":349674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL in macrophages affects myofibroblasts activities through PGE\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eA) The mRNA and protein expression of VEGF, MMP-9 in C57BL/6J myofibroblasts treated with MLKL\u003csup\u003e-/- \u003c/sup\u003eM1ø CM or with PGE\u003csub\u003e2\u003c/sub\u003e supplement. B) The mRNA and protein expression of VEGF, MMP-9 in C57BL/6J myofibroblasts treated with MLKL\u003csup\u003e-/- \u003c/sup\u003eM2ø CM or with PGE\u003csub\u003e2\u003c/sub\u003e supplement. GAPDH was used as a loading control (n = 3). Grayscale values were measured using ImageJ software. Results were expressed as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"Figure.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/eb898b651639eb14c83bd35a.jpg"},{"id":62960178,"identity":"5a431efc-4beb-4dbe-a03a-99054e8a25ef","added_by":"auto","created_at":"2024-08-21 13:12:21","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":577042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL in myofibroblasts affects macrophages activities through PGE\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eA) The production of IL-6, NO and TNF-α in C57BL/6J M1 macrophages after MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM treatment or MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM plus PGE\u003csub\u003e2\u003c/sub\u003e treatment.\u003cstrong\u003e \u003c/strong\u003eB) The production of IL-10, arginase and Ym1 in C57BL/6J M2ø after MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM treatment or MLKL\u003csup\u003e-/-\u003c/sup\u003e MFbCM plus PGE\u003csub\u003e2\u003c/sub\u003e treatment. GAPDH was used as a loading control (n = 3). Grayscale values were measured using ImageJ software. Results were expressed as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"figure.9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/3597291aa95a63f5b44c41e6.jpg"},{"id":62959441,"identity":"17f26336-de54-45c1-a824-cead3ef33e02","added_by":"auto","created_at":"2024-08-21 13:04:21","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2575126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLKL is involved in cutaneous wound healing and its roles in the interaction of M1/M2 macrophages and myofibroblasts in mice. \u003c/strong\u003eThe involvement of MLKL in the interaction of macrophages and fibroblasts, which might contribute to maintaining tissue repair microenvironment in skin wound site.\u003c/p\u003e","description":"","filename":"figure.10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/dfe695eef4e0bf63d84dbbb4.jpg"},{"id":81050869,"identity":"2e22924e-1133-4f8c-a306-8498bd6dab87","added_by":"auto","created_at":"2025-04-21 16:06:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23365930,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/ef4293ca-af2a-4fd2-99f1-d01cdadb394e.pdf"},{"id":62959439,"identity":"79bae04d-b9c1-49da-99f4-694ebc432a92","added_by":"auto","created_at":"2024-08-21 13:04:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2624984,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4576292/v1/ada200b6dbeed9747992a425.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MLKL are involved in the regulation of skin wound healing and the interplay between macrophages and myofibroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe skin, largest organ in the body, safeguards normal physiological activities and internal organs from environmental mechanical, microbial, or chemical stimuli. Constantly exposed to various external challenges, the skin frequently incurs wounds. An evolutionarily conserved process exists to close wounds, comprising hemostasis, inflammation, cellular proliferation, and remodeling \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Wound healing is typically regulated by a complex system of chemokines, cytokines, and tissue growth factors, alongside interactions between different cell types at the wound site \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Impairments or delays at any step in these processes can result in prolonged wound healing \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. A comprehensive understanding of the skin wound healing process at the molecular level is essential for enhancing therapeutic strategies for wound management.\u003c/p\u003e \u003cp\u003eSkin tissue regeneration requires the collaboration and crosstalk of various cell types and multiple mediators from the onset. After injury, macrophages are recruited to the wound site, following or simultaneously with neutrophil invasion \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Macrophages are highly efficient in tissue repair due to their well-known versatility and high plasticity \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. They phagocytose necrotic cellular debris and pathogenic material or microorganisms at the injury site through evolutionarily conserved receptors. Additionally, macrophages exhibit morphological changes and other actions in response to local signals \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Traditionally, macrophages are categorized into two main subsets: classically activated (M1 macrophages) and alternatively activated (M2 macrophages). M1 macrophages are induced by pro-inflammatory stimuli and further propagate inflammation by releasing inflammatory cytokines (e.g., IL-1, IL-6, and TNF-α). As wound healing progresses, these macrophages phagocytose apoptotic neutrophils, replacing them as the primary inflammatory cells. During the later stages of wound healing, M2 macrophages are responsible for releasing growth factors that promote angiogenesis, re-epithelialization, and fibroplasia \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The ablation of macrophages leads to delayed wound repair, directly evidencing their crucial role in wound healing \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Similarly, delayed re-epithelialization and angiogenesis are observed when macrophages are knocked down during early healing stages\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Macrophages in the wound bed can activate proliferation in fibroblasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. After injury, multiple subsets of fibroblasts become activated myofibroblasts, which contribute to tissue repair and scar formation \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Fibroblasts produce extracellular matrix (ECM) molecules that regulate tissue strength and resilience. An imbalance in ECM maintenance can lead to tissue dysfunction \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cross-talk between different cell types is crucial in the four overlapping phases of wound healing, though the underlying mechanisms are not yet fully elucidated. Cytokines are essential intermediates in the cross-talk between different cell types during wound healing. There are two primary modes of cytokine release: secretion from intact cells and extravasation from necrotic cells. Necroptosis, typically characterized by eventual cell lysis accompanied by the release of numerous cytokines or damage-associated molecular patterns (DAMPs), induces inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Necroptosis is abundant in wounds and is recognized as a significant factor affecting wound healing through the induction of inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Genetic studies have demonstrated that necroptosis is pro-inflammatory \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Necroptosis relies on the activity of the pseudokinase mixed lineage kinase domain-like (MLKL) protein \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Upon activation, MLKL forms oligomers and translocates to the plasma membrane, inducing cell death \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In mice, skin inflammation can be suppressed by the deletion of MLKL \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Receptor-interacting protein kinase 3 (RIPK3), the upstream regulator of MLKL, also contributes to the progression of wound healing\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Previous literature suggests that MLKL might play a role in tissue repair by regulating inflammation or other processes. However, direct evidence that MLKL engages in the skin wound healing process has yet to emerge.\u003c/p\u003e \u003cp\u003eIn the present study, transcriptome sequencing was employed to identify potential genes related to wound healing, with MLKL identified as a target in regulating this process. We used Wild-type (WT) C57BL/6J mice and MLKL-deficient (MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice as experimental models to examine the roles of MLKL in skin wound healing by creating dorsal cutaneous wounds. The roles of MLKL in the interaction between macrophages (M1 and M2) and myofibroblasts were investigated. Furthermore, we identified a potential intermediary, PGE\u003csub\u003e2\u003c/sub\u003e, responsible for mediating the roles of MLKL in regulating the interaction between M1/M2 macrophages and myofibroblasts.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome sequencing results: MLKL expression in skin wound site\u003c/h2\u003e \u003cp\u003eTo identify potential target genes associated with tissue repair, particularly those related to necrosis, we conducted transcriptome sequencing at the wound site of C57BL/6J mice from day 0 to day 14. Based on the transcriptome sequencing data, we selected genes associated with programmed cell death and mapped the top 30 upregulated genes. We identified four genes with sustained overexpression throughout the wound healing process from day 0 to day 14: Mlkl, Sycp2, Hk3, and Tnf (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequently, we used RT-PCR to assess MLKL mRNA expression levels during wound healing from day 0 to day 14. The RT-PCR results confirmed that MLKL overexpression was consistently observed in the wound tissue from day 0 to day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Together, these findings suggest that MLKL may serve as a potential regulator in the skin wound healing process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMorphological results: MLKL deficiency delays skin wound healing\u003c/h2\u003e \u003cp\u003eTo investigate the involvement of MLKL in skin wound healing in mice, the process of skin wound closure was compared between C57BL/6J mice and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice within 14 days. The results showed that delayed wound healing could be observed in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, as compared with C57BL/6J mice, which was obvious on 3rd, 5th, 7th, and 10th days \u003cb\u003e(Figure. 2A)\u003c/b\u003e. The data of wound area ratio was presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As shown in \u003cb\u003eFigure. 2B\u003c/b\u003e, the histological characteristics in the process of wound closure indicated that the healing speed of skin wound was impaired in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. On day 3, necrotic scab was observed on the surface of the wounds of both mice, with a large amount of serous fluid and cellulose exudation, and granulation tissue regeneration at the edge of the wounds. The difference was that the epidermis at the edge of the wound in C57BL/6J mice began to regenerate and grow into the wound, while this phenomenon did not occur at the edge of the wound in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. On day 5, the surface of wounds in both groups was covered with a thick layer of necrotic scabbed, and the granulation tissue filled the wounds and began to mature, with epidermal differentiation and thickening. The difference was that more granulation tissue was filled in the wounds of C57BL/6J mice than MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, and epidermal regeneration covered half of the wounds in C57BL/6J mice and 1/3 of the wounds in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. On day 7, the epidermis on the wound surface of the two kinds of mice regenerated completely, forming a thick layer covering the wound. The wound was filled with granulation tissue regeneration, and most of the granulation tissue was mature. The difference was that the granulation tissue near the regenerated epidermis in the wound of C57BL/6J mice was obviously congested. Between granulation tissue and epidermis regeneration in the wounds of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, local granulation tissue was congested and blood vessels were dilated. On day 10, the granulation tissue filled the wounds in the two kinds of mice, most of which were mature. The epidermis was completely regenerated and well differentiated, and the granulation tissue near the regenerated epidermis was obviously congested. The difference was that the collagen fibers in the wounds of C57BL/6J mice were significantly more than those of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. On day 12, granulation tissue filled the wounds of the two kinds of mice, and the epidermis regenerated completely and differentiated well to form thick epidermis. Most of the granulation tissue in the wounds of C57BL/6J mice was mature, with increased collagen fibers and local vascular congestion. Most granulation tissue in the wounds of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice began to mature, and scattered neutrophils were infiltrated in granulation tissue. On day 14, the granulation tissue in the wounds of both mice was filled and fully mature, and the epidermal regeneration completely covered the wounds to form a thick layer. These findings suggested that MLKL could be indispensable in re-epithelialization and wound contraction of skin wound healing in mice.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eCircular excision wound area (mm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e) on various days after injury\u003c/b\u003e Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences with \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). n\u0026thinsp;=\u0026thinsp;6 animals in each group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003eWound area (mm\u003csup\u003e2\u003c/sup\u003e)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC57BL/6J\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e24.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e25.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.63\u0026plusmn;***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMLKL deficiency decreases inflammatory response in wound tissue.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is known that MLKL regulates necrosis, and necrosis is closely related to inflammatory response in wound site after injury \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The necrosis related factors and inflammatory mediators was detected in wound tissue of C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The immunofluorescent staining showed that the protein expression of Caspase-3 and P53 in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound area was lower than in C57BL/6J mice \u003cb\u003e(Figure. 3A)\u003c/b\u003e. The protein expression of B Cell Leukemia 2 (Bcl-2) in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound area was higher than in C57BL/6J mice \u003cb\u003e(Figure.3A)\u003c/b\u003e. Above results indicated that MLKL deficiency impaired the cell death in wound area. Second, the inflammatory mediator including IL-6, TNF-α and prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e) secretion in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound tissue was lower than in C57BL/6J mice \u003cb\u003e(Figure. 3B)\u003c/b\u003e. For serum, IL-6 and TNF-α secretion in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound tissue was lower than in C57BL/6J mice. The PGE\u003csub\u003e2\u003c/sub\u003e in serum of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e was higher than that in C57BL/6J mice \u003cb\u003e(Figure. 3B)\u003c/b\u003e. Taken together, these results suggested that MLKL was involved in regulating necrosis and inflammatory response in wound healing process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMLKL deficiency impairs tissue growth factor expression in wound site\u003c/h2\u003e \u003cp\u003eTissue growth factors play a crucial role in wound healing. We examined the expression of growth factors, including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), estrogen receptor α (ERα), and matrix metalloprotein-9 (MMP-9), in the wound tissue of both C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Fluorescence staining revealed that the protein expression levels of EGF, VEGF, ERα, and MMP-9 were lower in the MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound area compared to C57BL/6J mice \u003cb\u003e(Figure. 4)\u003c/b\u003e. These findings suggest that MLKL is involved in regulating the synthesis of growth factors at the skin wound site.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMLKL in M1/M2 macrophages regulates the activity of myofibroblasts\u003c/h2\u003e \u003cp\u003eMacrophages activate myofibroblasts proliferation and its biological activity during the mid-stage of wound healing \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. We co-cultured M1\u0026oslash; CM withC57BL/6J myofibroblasts. The results indicated that M1 macrophages could induce ERα, VEGF and MMP-9 expression in myofibroblasts. To explore the roles of MLKL in M1 macrophages in regulating the activity of myofibroblasts, C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1\u0026oslash; CM was co-cultured withC57BL/6J myofibroblasts separately. We found that the inducible effect of M1\u0026oslash; CM on growth factors expression in myofibroblasts was inhibited when treatment replaced with MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003eM1\u0026oslash; CM \u003cb\u003e(Figure. 5A)\u003c/b\u003e. These results suggested that MLKL in M1 macrophages could regulate the growth factor expression in myofibroblasts. Similarly, we co-cultured M2\u0026oslash; CM withC57BL/6J myofibroblasts. The results demonstrated that M2\u0026oslash; CM could induce ERα, VEGF and MMP-9 expression in myofibroblasts. To explore the roles of MLKL in M2 macrophages in regulating the activity of myofibroblasts, C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M2\u0026oslash; CM was co-cultured with C57BL/6J myofibroblasts separately. We found that the inducible effect of C57BL/6J M2\u0026oslash; CM on growth factors expression in myofibroblasts was inhibited when treatment replaced with MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M2\u0026oslash; CM \u003cb\u003e(Figure. 5B)\u003c/b\u003e. These results suggested that MLKL in M2 macrophages could regulate the growth factor expression in myofibroblasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMLKL in fibroblasts regulate the activity of M1/M2 macrophages\u003c/h2\u003e \u003cp\u003eTo investigate the effects of myofibroblasts on the activity of macrophages. We co-cultured C57BL/6J MFbCM with M1 and M2 macrophages separately. The results indicated that C57BL/6J MFbCM induced the synthesis of IL-6, nitric oxide (NO), and TNF-α in M1 macrophages, whereas this inductive effect was inhibited when MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM was used instead \u003cb\u003e(Figure. 6A)\u003c/b\u003e. This suggests that MLKL in myofibroblasts regulates the activity of M1 macrophages. Ym1 and arginase are primarily expressed in macrophages at the site of injury \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We observed lower expression levels of arginase and Ym1 in the MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e skin wound site compared to the C57BL/6J wound site \u003cb\u003e(Figure. 6C)\u003c/b\u003e. Additionally, the results showed that C57BL/6J MFbCM induced the synthesis of IL-10, arginase, and Ym1 in M2 macrophages, whereas this inductive effect was inhibited when MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM was used \u003cb\u003e(Figure. 6B)\u003c/b\u003e. This suggests that MLKL in myofibroblasts also regulates the activity of M2 macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePGE\u003csub\u003e2\u003c/sub\u003e is one of the mediators in the cross-talk between macrophages and myofibroblasts\u003c/h2\u003e \u003cp\u003eThe results above indicate that MLKL plays a role in the interaction between macrophages and myofibroblasts. We observed that the expression of cyclooxygenase-2 (COX-2) and the secretion of PGE\u003csub\u003e2\u003c/sub\u003e were lower in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1/ M2 macrophages and myofibroblasts compared to C57BL/6J cells \u003cb\u003e(Figure. 7)\u003c/b\u003e, suggesting that PGE\u003csub\u003e2\u003c/sub\u003e might serve as an intermediary in the interaction between macrophages and myofibroblasts. To explore this further, we supplemented MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM or M1\u0026oslash; / M2\u0026oslash; CM with exogenous PGE\u003csub\u003e2\u003c/sub\u003e to match the amount lost in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells. The results demonstrated that MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1\u0026oslash; CM or M2\u0026oslash; CM supplemented with PGE\u003csub\u003e2\u003c/sub\u003e enhanced the expression of VEGF and MMP-9 in myofibroblasts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. However, when MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM was supplemented with PGE\u003csub\u003e2\u003c/sub\u003e, the effects differed: IL-6 synthesis remained unaffected, NO secretion was further inhibited, and TNF-α was enhanced in M1 macrophages \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Additionally, MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM supplemented with PGE\u003csub\u003e2\u003c/sub\u003e decreased IL-10 and Ym1 expression, while enhancing arginase expression in M2 macrophages \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Therefore, these findings suggest that the roles of PGE\u003csub\u003e2\u003c/sub\u003e in inducing M1/M2 macrophage activation by myofibroblasts are more complex than those in inducing myofibroblast activation by M1/M2 macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMLKL-dependent necrosis is commonly associated with adverse pathologies; however, our study reveals that MLKL is essential for normal skin wound healing. \u003cem\u003eIn vivo\u003c/em\u003e experiments demonstrate that MLKL deficiency results in delayed wound repair. While MLKL is traditionally thought to affect wound healing through modulation of necrosis-induced inflammation, our findings suggest that its role extends beyond this mechanism. Firstly, MLKL overexpression persists throughout the entire wound healing process, from incision to closure, rather than being restricted to the inflammation phase. Secondly, MLKL deficiency negatively impacts both the wound healing rate and the expression of growth-related factors. Thirdly, the regulatory effect of macrophages on myofibroblast activity is diminished in the absence of MLKL. Conversely, while myofibroblasts can activate macrophages, this activation is partially inhibited by MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e myofibroblasts. Our study also establishes that PGE\u003csub\u003e2\u003c/sub\u003e functions as an intermediary in the interaction between macrophages and myofibroblasts. In summary, MLKL plays a crucial role in skin wound healing by mediating a complex network of cellular interactions and cytokine expressions, thereby contributing to the establishment of a tissue repair microenvironment at the wound site.\u003c/p\u003e \u003cp\u003eThe initial damage stimulus induces a conformational change in cells at the wound site, leading to plasma membrane translocation and the lethal permeation of the lipid bilayer. This process results in the release of cellular contents and the subsequent activation of an inflammatory response \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Pro-inflammatory chemokines and cytokines released by activated macrophages, primarily M1 macrophages, initiate the recruitment and activation of additional immune cells, including neutrophils and bone marrow-derived monocytes, to the injury site. These immune cells further amplify inflammation to eliminate damage signals and necrotic cells \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Necroptosis is mediated by RIPK3 \u003csup\u003e19\u003c/sup\u003e. RIPK3 deficiency significantly delays wound closure and impairs wound healing quality, as evidenced by delayed re-epithelialization, angiogenesis, granulation tissue formation, and collagen deposition \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. MLKL, a functional substrate for RIPK3, acts as an adaptor protein in necrosis signal transduction \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Both RIPK3 and MLKL overexpression are observed at early stages of skin wound healing. Additionally, three other targets (Sycp2, HK3 and TNF-α) are overexpressed at the wound site. Sycp2, a synaptonemal complex protein, is associated with meiosis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. HK3 is essential for initiating glycolysis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Both Sycp2 and HK3 may be involved in cellular differentiation related to inflammatory and immune responses \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. TNF-α is crucial in both apoptosis and necroptosis, and its interaction with MLKL in necroptosis is well-documented \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, the co-overexpression of TNF-α and MLKL could be observed at wound sites as expected. In MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003emice, delayed skin wound closure and impaired morphological characteristics throughout the healing process are observed. Additionally, the concentration of inflammatory cytokines, including PGE\u003csub\u003e2\u003c/sub\u003e, TNF-α, and IL-6, is lower in the wound tissue of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to C57BL/6J mice three days post-injury. Serum levels of TNF-α and IL-6 are also reduced in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, while PGE\u003csub\u003e2\u003c/sub\u003e levels are elevated in their serum after injury. The wound site data are likely more indicative of the local inflammatory microenvironment than serum data, which reflects a systemic rather than a localized response \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. MLKL may reduce the inflammatory response at the wound site through two potential mechanisms: it could directly regulate the synthesis of inflammatory cytokines, leading to decreased intracellular cytokine production in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Alternatively, reduced cell death, as evidenced by altered expression of P53, Bcl-2, and Caspase-3, could result in less cellular content release, potentially reducing inflammatory cytokine release in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound sites. It is possible that both mechanisms contribute to the reduced inflammatory cytokine release observed in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e skin wounds. Overall, these results suggest that MLKL deficiency impairs skin wound healing, likely due to diminished inflammatory responses at the wound site, particularly during the early stages of healing.\u003c/p\u003e \u003cp\u003eAs wound healing progresses from the pro-inflammatory to the pro-healing phase, it transitions into the remodeling stage. This stage is marked by a shift in macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The debate continues on whether M2 macrophages at injury sites arise from blood monocytes or result from the phenotypic conversion of M1 macrophages \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These anti-inflammatory macrophages are crucial for inflammation resolution and the progression of remodeling. They contribute to the healing process through their regenerative properties by secreting angiogenic and growth factors, cytokines, and chemokines, such as metalloproteinases (MMPs), VEGF, IL-8, TGF-β, IL-10, and arginase, which are essential for recruiting and activating other cells. Depletion of macrophages during the regenerative phase can directly or indirectly impact wound revascularization, matrix production, and re-epithelialization \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our study reveals that MLKL and RIPK3 are sustained in the wound area during mid- and late-stages of healing, indicating that RIPK3-MLKL signaling may be important not only in the early inflammatory phase but also in the later stages of wound repair. We found that MLKL deficiency impairs the expression of growth-related factors, such as EGF, VEGF, MMP-9, and ERα, during the mid- and late-stages of wound healing. This suggests that MLKL plays a role in regulating growth factor expression at the wound site during the later stages of healing. The delayed wound regeneration observed in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, as evidenced by H\u0026amp;E staining, aligns with these findings. Specifically, collagen fibers and mature granulation tissue were significantly more abundant in wounds of C57BL/6J mice compared to MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice at later stages of healing.\u003c/p\u003e \u003cp\u003eMiscommunication between macrophages and fibroblasts is recognized as a critical factor that can shift the balance from physiological repair to pathological fibrosis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. It is well-documented that macrophage depletion during wound healing leads to a reduction in fibroblast numbers and that macrophages play a role in scavenging TGF-β, a key regulator of fibroblast/myofibroblast activation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Supernatants from C57BL/6J M1 macrophages were found to enhance the expression of VEGF and MMP-9 in fibroblasts. This effect was partially inhibited when supernatants from MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1 macrophages were used instead, indicating that MLKL in macrophages may regulate myofibroblast activity. Additionally, PGE\u003csub\u003e2\u003c/sub\u003e synthesis, which is known to increase at both early and late stages of wound healing \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We hypothesized that PGE\u003csub\u003e2\u003c/sub\u003e is a potential mediator in the cross-talk between M1 macrophages and myofibroblasts, as we found that COX-2 (the upstream enzyme of PGE\u003csub\u003e2\u003c/sub\u003e synthesis) expression and PGE\u003csub\u003e2\u003c/sub\u003e levels in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound tissue were lower than in C57BL/6J wound tissue \u003cb\u003e(Figure. 7)\u003c/b\u003e. To investigate this further, we replenished PGE\u003csub\u003e2\u003c/sub\u003e in a co-culture system of MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1 macrophages and myofibroblasts. This restoration of PGE\u003csub\u003e2\u003c/sub\u003e led to a recovery in the expression patterns of VEGF and MMP-9 in myofibroblasts. Conversely, MLKL in myofibroblasts was found to regulate the synthesis of IL-6, NO, and TNF-α in M1 macrophages through PGE\u003csub\u003e2\u003c/sub\u003e. These findings suggest that PGE\u003csub\u003e2\u003c/sub\u003e may act as an intermediary in the MLKL-mediated interaction between M1 macrophages and myofibroblasts. Regarding the mechanism behind the reduced PGE\u003csub\u003e2\u003c/sub\u003e concentration in supernatants from MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e macrophages (both M1 and M2) and myofibroblasts, two potential explanations exist: MLKL deficiency might directly reduce PGE\u003csub\u003e2\u003c/sub\u003e synthesis in these cells, or it might delay membranolysis, trapping PGE\u003csub\u003e2\u003c/sub\u003e within the cells. Our data, particularly the reduced COX-2 expression in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells, support the first possibility. Taken together, MLKL may play a crucial role in maintaining the early-stage inflammatory microenvironment at the wound site by modulating the interaction between M1 macrophages and fibroblasts.\u003c/p\u003e \u003cp\u003eM2 macrophages produce anti-inflammatory cytokines and growth factors that mitigate inflammation, activate fibroblasts, and promote angiogenesis and wound contraction \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. It has been demonstrated that ablation of macrophages reduces myofibroblast numbers in the wound bed, impairs myofibroblast function, and hinders wound healing \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Prolonged pathological stimulation leading to unresolved crosstalk between M2 macrophages and fibroblasts often results in pathological fibrosis or chronic wounds \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, understanding the interaction between M2 macrophages and fibroblasts is essential. We observed that the expression of growth factors EGF, VEGF, and MMP-9 peaks at the late stage of wound healing; however, their levels were lower in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e wound sites compared to C57BL/6J sites. MLKL in M2 macrophages appears to regulate VEGF and MMP-9 expression in myofibroblasts, suggesting that MLKL deficiency might lead to reduced growth factor expression at the wound site. Additionally, MLKL in fibroblasts can regulate the expression of arginase, Ym1, and IL-10 in M2 macrophages, indicating that MLKL in fibroblasts may also influence M2 macrophage activity \u003cb\u003e(Figure. 6)\u003c/b\u003e. Ym1 and arginase, which are predominantly expressed in macrophages at injury sites, play crucial roles in wound healing and resolution \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Arginase contributes to wound healing through the production of L-ornithine, with its absence impairing cutaneous wound healing and its overproduction contributing to fibrosis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. IL-10 promotes the differentiation of macrophages towards pro-regenerative phenotypes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. PGE\u003csub\u003e2\u003c/sub\u003e released from myofibroblasts, along with its receptor agonist, enhances arginase activity in M2 macrophages, further supporting the role of PGE\u003csub\u003e2\u003c/sub\u003e in regulating M2 macrophage activity \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We also found reduced COX-2 and PGE\u003csub\u003e2\u003c/sub\u003e synthesis in MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M2 macrophages. Similar to its role in the interaction between M1 macrophages and myofibroblasts, PGE\u003csub\u003e2\u003c/sub\u003e is involved in the interaction between M2 macrophages and myofibroblasts. These findings highlight the importance of MLKL and PGE\u003csub\u003e2\u003c/sub\u003e in mediating the complex interactions between macrophages and fibroblasts during wound healing \u003cb\u003e(Figure. 10)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides direct evidence that MLKL plays a crucial role in skin wound healing by modulating the wound site's microenvironment. We have also elucidated the involvement of MLKL in the interaction between macrophages and fibroblasts, identifying PGE\u003csub\u003e2\u003c/sub\u003e as a potential intermediary in the MLKL-mediated communication between both M1 and M2 macrophages and fibroblasts.\u003c/p\u003e"},{"header":"Material \u0026 Method","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe mice experiment of present study were performed under the guidance of the Regulations for the Administration of Affairs Concerning Experimental Animals in China (2017). The experimental design was approved by the Animal Welfare and Research Ethics Committee of the university (approval ID: NND2022021). C57BL/6J mice were provided by the Model Animal Research Center of Nanjing University, Nanjing, China. MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were gifted by Professor Shuixing Yu of Inner Mongolia Agricultural University, who has previously done related research\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The mice were housed in a clean environment (12 h day/night cycles, 22\u0026ndash;24 ℃ and 50% humidity) supplied with unlimited food and water. Mice at age eight-weeks-old (25-30g) were used in all experiments. This study was conducted in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e study, transcriptome sequencing and analysis of wound sites from C57BL/6J mice were conducted to identify potential genes involved in skin wound healing (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). To investigate the roles of MLKL in this process, we created skin wounds in both C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The wound healing ratio, morphological characteristics, and expression of tissue repair-related factors at the wound site were monitored throughout the healing process using H\u0026amp;E staining and immunofluorescence staining (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e study, M1/M2 macrophages and myofibroblasts were used to investigate the roles of MLKL in the interaction between these cell types. We co-cultured M1 macrophage conditioned medium (M1\u0026oslash; CM) or M2\u0026oslash; CM withC57BL/6J myofibroblasts as previously described \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The expression of ERα, VEGF, and MMP-9 in myofibroblasts was detected. Further, to explore the roles of MLKL in M1/M2 macrophages in regulating myofibroblast activity, C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M1\u0026oslash; CM (or MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e M2\u0026oslash; CM) were co-cultured withC57BL/6J myofibroblasts separately. The expression of ERα, VEGF, and MMP-9 in myofibroblasts was assessed by Western blotting and RT-PCR. To investigate the effects of myofibroblasts on M1/M2 macrophages, we co-cultured myofibroblast conditioned medium (MFbCM) with M1/M2 macrophages separately. The expression of IL-6, nitric oxide (NO), and TNF-α in M1 macrophages was measured, as well as IL-10, arginase, and Ym1 in M2 macrophages. Additionally, to explore the roles of MLKL in myofibroblasts in regulating the activity of M1/M2 macrophages, C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MFbCM were co-cultured with M1/M2 macrophages separately. The expression of IL-6, NO, TNF-α and IL-10 was detected by ELISA, arginase and Ym1 expression in M2 macrophages was detected by Western blotting. Finally, the roles of PGE\u003csub\u003e2\u003c/sub\u003e in the MLKL-mediated interaction between macrophages and myofibroblasts were investigated. The experimental design abstract is attached (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCutaneous wound model\u003c/h2\u003e \u003cp\u003eThe circular excisional wound was directly created on the dorsal skin of C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Briefly, the experimental mice were anesthetized with isoflurane inhalation. The dorsum was shaved and sterilized with 75% alcohol. A 6-mm diameter skin biopsy punch was used to create an excision wound extending to the fascia. Wound closure progression was measured daily until day 14 post-wound. At days 0, 3, 5, 7, 10, 12, and 14 after wound injury, mice were sacrificed following anesthesia, and skin samples were collected from the entire wound sites (including the scab and epithelial margins). The skin samples were fixed and stored in 10% formalin for histological analysis or frozen in liquid nitrogen and stored at -80\u0026deg;C for molecular experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis\u003c/h2\u003e \u003cp\u003eTotal mRNA was extracted using TRIzol reagent (Invitrogen, CA, USA). Libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Differentially expressed genes (DEGs) were identified using DESeq2 and screened under the criteria of |log2 Fold Change| \u0026gt; 1.5 and \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The identified DEGs underwent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using OECloud tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com/task/\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com/task/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Significant enrichment was defined by a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and the top 30 enrichment results are presented.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWound healing rate determination\u003c/h2\u003e \u003cp\u003eThe wound healing rate at 0, 3, 5, 7, 10, 12, and 14 days was calculated using the wound surface area compared to the original wound area. The percentage of wound closure was determined using the formula: (AreaDay 0-AreaDay)/AreaDay 0\u0026times;100%. Digital images of the wound surface were taken using a SONY Alpha 6400 camera. The wound area was quantified using ImageJ software (National Institutes of Health, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eThe skin tissue samples fixed in 10% formalin were sectioned into 2 \u0026micro;m in thickness. The sections were dehydrated using a graded series of ethanol (100%, 75%, 50%, and 25%), embedded in paraffin, and stained with hematoxylin and eosin (H\u0026amp;E). Images of the stained sections were acquired using an Axio Scan.Z1 slide scanner (Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe excised dorsal tissue samples from C57BL/6J mice and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were frozen in liquid nitrogen and stored at -80\u0026deg;C. Subsequently, the tissues were thawed and embedded in Tissue-Tek OCT compound (Sakura Finetek, CA, USA), and serial cryosections of 6 \u0026micro;m thickness were prepared using a freezing microtome. The sections were fixed in cold acetone for 10 minutes, washed with cold endotoxin-free phosphate-buffered saline (PBS) containing 0.25% Tween-20, and blocked for 1 hour with 3% bovine serum albumin. Primary antibodies were then added, and the sections were incubated in the dark for 14 hours at 4\u0026deg;C. After incubation, the slides were washed three times in PBS with 0.25% Tween-20 and incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature. DAPI (4',6-diamidino-2-phenylindole) was used for nuclear counterstaining. Fluorescence signals were captured using a confocal microscope (LSM 800, Zeiss, Oberkochen, Germany). Fields were selected randomly for imaging. The details of the antibodies used are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAntibodies used in immunofluorescence staining\u003c/b\u003e p53: Tumor protein 53; Bcl-2: B-cell lymphoma 2; Caspase-3: Cysteinyl aspartate specific proteinase 3; ERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; EGF: Epidermal growth factor; VEGF: Vascular endothelial growth factor; Ym1: Chitinase-3-like Protein 1; Dnk: Donkey; pAb: polyclonal antibody; Rb: Rabbit.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCat No.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2524T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBcl-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15071\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eabcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab49822\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGeneTex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGTX13538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20ug/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNovus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNBP2-41233\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eabcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab184266\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200ug/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSanta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSC-7269\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003earginase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSanta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSC-47715\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYm1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStemcell technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDnk pAb to Rat IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eabcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab150153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat pAb to Rb IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eabcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAb150079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of bone marrow-derived macrophages (BMDMs)\u003c/h2\u003e \u003cp\u003eThe BMDMs were isolated from murine femurs and tibias, cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, Excell Bio, Shanghai, China), 1.2% Glutamine (Thermo scientific, Rockford, IL, USA), 2.4% penicillin-streptomycin (Gibco, USA), combined with 20 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech, NJ, USA) at a density of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in a 6-well plate. The culture medium was replaced at 24 h interval up to 5 days. The BMDMs were treated with 1ug /mL LPS (PeproTech, NJ, USA) for 24 h to differentiate into M1 macrophages, which was identified by flow cytometry (Figure. S2 in Supplementary Material). The BMDMs were treated with 20 ng/mL IL-4 in combination with 20 ng/mL IL-13 (PeproTech, NJ, USA) for 48 h to differentiate into M2 macrophages, which was identified by flow cytometry (Figure. S2 in Supplementary Material). The M1 and M2 macrophages conditioned medium (M1\u0026oslash; CM and M2\u0026oslash; CM) were collected for further experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMice skin myofibroblasts cultivation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt first, the fibroblasts were isolated from C57BL/6J and MLKL\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. In brief, the ear explants were minced and incubated in 0.25% trypsin-EDTA (Hyclone, UT, USA) for 1 h at 37\u0026deg;C to remove the epidermis. The obtained explants were placed in tissue culture plates, and migration of fibroblasts from tissue explant to dish could be observed within 2\u0026ndash;5 days. After 2 weeks, tissue explants were removed, and fibroblasts were re-seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in a 6-well plate and cultured in DMEM/F12 medium supplemented with 10% FBS and 2% penicillin-streptomycin combined with 100 pg/mL transforming growth factor-β (TGF-β, PeproTech, NJ, USA). The medium was replaced every 24 h until the fifth day when the fibroblasts were induced to myofibroblasts. Subsequently, the culture medium was replaced with DMEM/F12 medium supplemented with 10% FBS and 2% penicillin-streptomycin, and cultured for 2 days to remove TGF-β. The myofibroblasts were identified by α-smooth muscle actin (SMA) expression using immunofluorescence assay (Figure. S3 in Supplementary Material). The myofibroblasts conditioned medium (MFbCM) was collected for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis (WB)\u003c/h2\u003e \u003cp\u003eFor total cellular protein extraction, cells were treated with the Mammalian Protein Extraction Reagent (Thermo scientific, Rockford, IL, USA). The protein concentrations in the samples were quantified using the BCA Assay Kit (Thermo scientific, Rockford, IL, USA). For Western blot analysis, 10 \u0026micro;g of total protein per lane were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. Protein bands were visualized using the Chemiluminescent Substrate (Thermo scientific, Rockford, IL, USA). Grayscale values were quantified using ImageJ software (National Institutes of Health, MD, USA). Primary antibodies, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAntibodies used in western blot\u003c/b\u003e ERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; Ym1: Chitinase-3-like Protein 1; COX-2, Cyclooxygenase-2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCat No.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.4 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGeneTex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGTX13538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 ug/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNovus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNBP2-41233\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 ug/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNovus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNB100-2381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003earginase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSC-47715\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYm1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStemcell technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit polyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAffinity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAF7021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOX-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit monoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70 KDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCell signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD5H5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat Anti-Rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(H\u0026thinsp;+\u0026thinsp;L) HRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAffinity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat Anti-Mouse IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(H\u0026thinsp;+\u0026thinsp;L) HRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAffinity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA) analysis\u003c/h2\u003e \u003cp\u003eThe harvested wound tissue (0.2\u0026ndash;0.4 g) was cut into pieces, homogenized and lysed with T-PERTM Tissue Protein Extraction Reagent (Thermo scientific, Rockford, IL, USA). The blood was gained from the eyeballs and centrifuged at 3000 \u0026times; g for 10 min to extract serum. The concentrations of IL-6 (Biolegend, CA, USA), TNF-α (Biolegend, CA, USA) and PGE\u003csub\u003e2\u003c/sub\u003e (Cayman Chemical, Ann Arbor, MI) were measured in the tissue extracts and serum. The concentration of IL-6, TNF-α, IL-10 (Invitrogen, Thermo Fisher Scientific, CA, USA) and NO (Beyotime, Shanghai, China) supernatants of cultured macrophages was measured according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eReal-time RT-PCR analysis\u003c/h2\u003e \u003cp\u003eThe total mRNA was extracted using the Total RNA Miniprep Kit (Axygen, California, USA) from cultured cells. The extracted RNA was reverse-transcribed into cDNA by a cDNA Reverse Transcription Kit (Vazyme, Nanjing, China). Real-time PCR reactions were conducted using the ABI QuantStudio 7 (Thermo Scientific, IL, USA). The PCR protocol was as follows: initial denaturation for 30 seconds at 95\u0026deg;C, followed by 35 cycles of 5 seconds at 95\u0026deg;C (denaturation), 34 seconds at 60\u0026deg;C (annealing), and 20 seconds at 72\u0026deg;C (elongation). GAPDH served as an internal control. Results are presented as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e (where ΔΔCt\u0026thinsp;=\u0026thinsp;ΔCt \u0026ndash; ΔCt control and ΔCt\u0026thinsp;=\u0026thinsp;Ct target -Ct GAPDH). The primers used in RT-PCR are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_008084.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward:5' -AGGTCGGTGTGAACGGATTTG-3'\u003c/p\u003e \u003cp\u003eReverse:3' -GGGGTCGTTGATGGCAACA-5\u0026rsquo;'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_007956.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eERα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward:5'- TTCTCCCTTTGCTACGTCAC-3'\u003c/p\u003e \u003cp\u003eReverse:3'- ATCGCTTTGTCAACGACTTC-5'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_013599.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMMP-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward:5'- CGCCTTGGTGTAGCACAACA-3'\u003c/p\u003e \u003cp\u003eReverse:3'- ACAGGGTTTGCCTTCTCCGTT-5'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_009505.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward:5'- CTGTAACGATGAAGCCCTGGAG-3'\u003c/p\u003e \u003cp\u003eReverse:3'- TGGTGAGGTTTGATCCGCAT-5'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_029005.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMLKL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward:5'- TATGTCTCCCCTGAGAGACTGAAAA-3'\u003c/p\u003e \u003cp\u003eReverse:3'- TTCCCAGAGTACAATTCCAAAGCTA-5'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eERα: Estrogen receptors α; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; MLKL: mixed lineage kinase domain-like protein.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using GraphPad Prism 8 (GraphPad Software InC, USA) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple-comparisons test or two-way ANOVA with Bonferroni\u0026rsquo;s post-hoc test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Jinling Wang and Dr. Yulin Ding for their kind assistance in H\u0026amp;E staining. Graphic abstract of present study created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuangyi Zhang, Jiamin Zhao, Bo Liu and Surong Hasi contributed to the study conception and design. Material preparation, data collection and analysis were performed by Jiamin Zhao, Yunjie Bai, Zhiguo Gong, Zhuoya Yu, Wenhui Bao and Wei Mao. The first draft of the manuscript was written by Jiamin Zhao and all authors commented on previous versions of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data for this study are available by contacting the corresponding authors upon reasonable request. The sequencing data of this study are openly available in GenBank of NCBI (accession No. GSE273056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2024MS03064), National Natural Science Foundation of China (32202879), Research Program of Science and Technology at Universities of Inner Mongolia Autonomous Region (NJZZ23035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing financial interests or personal relations that might have influenced the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed according to the regulations of the Administration of Affairs Concerning Experimental Animals in China. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of the Inner Mongolia Agricultural University (approval ID: NND2022021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRaziyeva, K. \u003cem\u003eet al.\u003c/em\u003e Immunology of Acute and Chronic Wound Healing. \u003cem\u003eBiomolecules\u003c/em\u003e 11(5), 700\u0026ndash;700 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodwin, A. \u003cem\u003eet al.\u003c/em\u003e Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing. Plos One 10(10), e0140514 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkinson, H. N. \u0026amp; Hardman, M. J. 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Y. \u0026amp; Mongeau, L. Immunomodulatory Microgels Support Proregenerative Macrophage Activation and Attenuate Fibroblast Collagen Synthesis. Advanced Healthcare Materials 11(11), e2102366 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernando, M. R., Giembycz, M. A. \u0026amp; McKay, D. M. Bidirectional crosstalk via IL-6, PGE\u003csub\u003e2\u003c/sub\u003e and PGD\u003csub\u003e2\u003c/sub\u003e between murine myofibroblasts and alternatively activated macrophages enhances anti-inflammatory phenotype in both cells. British Journal of Pharmacology 173(5), 899\u0026ndash;912 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, S.X. \u003cem\u003eet al.\u003c/em\u003e Non-Hematopoietic MLKL Protects Against Salmonella Mucosal Infection by Enhancing Inflammasome Activation. Frontiers in Immunology 9, 119 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Skin, Woud healing, MLKL, Macrophages, Myofibroblasts","lastPublishedDoi":"10.21203/rs.3.rs-4576292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4576292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pseudokinase mixed lineage kinase domain-like protein (MLKL) acts as a crucial effector in the necroptosis pathway. It is widely recognized that MLKL-dependent necrosis is closely related to inflammation in wound healing, which contributes to detrimental pathologies. In present study, transcriptome sequencing data indicate sustained overexpression of MLKL throughout the wound healing process, extending beyond the early inflammation phase. \u003cem\u003eIn vivo\u003c/em\u003e experiments clearly demonstrate that MLKL deficiency delays skin wound healing, as evidenced by morphological observations and pathological characteristics. MLKL deficiency impairs the synthesis of inflammatory factors (IL-6, TNF-α, and PGE\u003csub\u003e2\u003c/sub\u003e) and tissue repair-related molecules (EGF, VEGF, ERα, and MMP-9) at the wound site, potentially leading to delayed wound closure. Furthermore, we have identified the roles of MLKL and PGE\u003csub\u003e2\u003c/sub\u003e in the interaction between macrophages (both classically activated and alternatively activated) and myofibroblasts, an interaction essential at each stage of wound healing. Our findings suggest that MLKL's involvement in wound healing may not solely rely on necrosis-induced inflammatory responses during the early stages but also contributes to other activities in tissue regeneration.\u003c/p\u003e","manuscriptTitle":"MLKL are involved in the regulation of skin wound healing and the interplay between macrophages and myofibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 13:04:15","doi":"10.21203/rs.3.rs-4576292/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-25T06:43:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-24T17:14:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70696291168896205326154400377653751536","date":"2024-10-11T08:02:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T02:32:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7772613353839002441911186957657505535","date":"2024-08-08T12:25:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53857961362998533170753673962983947159","date":"2024-08-08T12:25:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T11:07:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-08T11:06:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-29T03:47:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-25T12:30:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-13T12:43:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fcdde61-f2df-4b88-b135-180e07fb7655","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36267211,"name":"Biological sciences/Immunology"},{"id":36267212,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-04-21T16:00:56+00:00","versionOfRecord":{"articleIdentity":"rs-4576292","link":"https://doi.org/10.1038/s41598-025-97729-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-19 15:57:36","publishedOnDateReadable":"April 19th, 2025"},"versionCreatedAt":"2024-08-21 13:04:15","video":"","vorDoi":"10.1038/s41598-025-97729-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-97729-2","workflowStages":[]},"version":"v1","identity":"rs-4576292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4576292","identity":"rs-4576292","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}